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A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore
Abstract
We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins-Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1-as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein-DNA conformation that evokes the active centromere.
... Mutation in MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and leads to suppression of temperature sensitivity and DNA replication defects in a cdc7-7 strain (Hardy et al., 1997;Sclafani et al., 2002;Hoang et al., 2007). Studies to date have defined one kinetochore substrate for Cdc7 namely Ctf19 (Hinshaw et al., 2017), a component of the COMA (Ctf19, Okp1, Mcm21, and Ame1) complex (Ortiz et al., 1999). Cdc7-mediated phosphorylation of Ctf19 is required for its association with cohesin loaders Scc2/4, and the loading of cohesin to the CEN chromatin (Hinshaw et al., 2017). ...
... Cdc7-mediated phosphorylation of Ctf19 is required for its association with cohesin loaders Scc2/4, and the loading of cohesin to the CEN chromatin (Hinshaw et al., 2017). Interestingly, Ctf19 interacts with Cse4, and this interaction is important for the recruitment of the COMA complex to CEN chromatin and maintenance of the functional integrity of the kinetochore (Ortiz et al., 1999). A potential role for Cdc7-mediated phosphorylation of Cse4 in kinetochore function and chromosome segregation has not been characterized. ...
... DDK enrichment has been observed at the budding yeast kinetochores (Natsume et al., 2013;Hinshaw et al., 2017), and proposed to regulate loading of CEN cohesion through phosphorylation of Ctf19 (Hinshaw et al., 2017). Moreover, Ctf19 as well as other members of the COMA complex interact with Cse4, which is required for the CEN recruitment of COMA complex and kinetochore assembly (Ortiz et al., 1999). Therefore, we examined whether Cdc7 kinase interacts with and phosphorylates Cse4 in vitro. ...
Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN) which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores and defects in chromosome segregation are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase dead variant of Cdc7 ( cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phosphodeficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.
... Mif2 (Ortiz et al., 1999). Other subunits were found by genetic lethality screens, affinity purification and mass spectrometry and a variety of subcomplexes were successfully reconstituted (Hyland et al., 1999, Measday et al., 2002, De Wulf et al., 2003, Hornung et al., 2011, Hornung et al., 2014, Schmitzberger and Harrison, 2012, Schmitzberger et al., 2017, Weir et al., 2016. ...
... Most of the Ctf19 complex subunits are conserved to the CCAN of higher eukaryotes and they also share most of their functionality. One known exception is the involvement of the CCAN in CENP-A loading in higher eukaryotes, as all known Ctf19 subunits are dependent on prior CBF3, Mif2 and Cse4 localisation to the centromere (Ortiz et al., 1999, Hyland et al., 1999, De Wulf et al., 2003. ...
... The centromere specific nucleosome is then recognised and bound by Mif2 CENP-C which acts as a central platform to recruit other kinetochore proteins (Carroll et al., 2010, Kato et al., 2013. Besides Mif2, some subunits of the Ctf19 CCAN complex also recognise and interact with the centromere-specific nucleosome, a conserved interaction important for Ctf19 CCAN stability at the centromere (Ortiz et al., 1999, De Wulf et al., 2003. Furthermore, Mif2 directly interacts with components of the Ctf19 and MIND complexes (Cohen et al., 2008, Hornung et al., 2014. ...
Kinetochores connect centromeres with spindle microtubules during mitosis and meiosis to ensure correct chromosome segregation. The initial step of kinetochore establishment in point centromere species, like budding yeast, is the sequence-specific recognition of the centromere by the essential CBF3 complex. CBF3 consists of four proteins, Ndc10, Cep3, Ctf13 and Skp1, and its binding to the centromere is required for the assembly of all other kinetochore proteins. Beside this, the complex is involved in the recruitment of the centromere-specific histone Cse4 through its interaction with the histone chaperone Scm3 and it also impacts the overall conformation of the centromere through DNA looping and bending. Besides its crucial role in kinetochore establishment, little structural data is available for the CBF3 complex, mainly due to difficulties with recombinantly expressing and purifying the full complex. This thesis describes a working co-expression and purification protocol for CBF3, which allowed structural as well as functional studies of the complex and led to the cryoEM structure of the core CBF3 complex, comprising the centromere-binding Cep3 and the regulatory subunits Ctf13 and Skp1. Besides the overall core architecture, this structure provides the first insights into the inherently unstable subunit Ctf13, as well as a potential new conformation of the Skp1/F-box interaction. Furthermore, it provides interesting insights into the unusual DNA-binding properties of the dimeric Cep3 to a single consensus site in the centromere, a matter of some debate in the field. Biochemical studies revealed a potential and previously undescribed regulatory mechanism of DNA-binding activity through phosphorylation of the Skp1 subunit. Additional work was undertaken to better understand how Ndc10 binds to the core complex and therefore how the full CBF3 assembles, as well as interaction studies with the centromere-specific nucleosome. As a side project, interaction studies between Ndc10 and Scm3 were undertaken, to elucidate the proposed Cse4-loading function of CBF3.
... The COMA complex represents a subassembly within the Ctf19 complex, and it links the inner kinetochore to the MIND complex (De Wulf et al, 2003). COMA was named for the S. cerevisiae components Ctf19 CENP-P (Hyland et al, 1999), Okp1 CENP-Q (Ortiz et al, 1999), Mcm21 CENP-O (Poddar et al, 1999) and Ame1 CENP-U (Burns et al, 1994;Cheeseman et al, 2002;Gavin et al, 2002; superscript indicates the mammalian orthologs). The mammalian equivalent of yeast COMA is the CENP-O/P/Q/U complex (Musacchio & Desai, 2017). ...
... Apart from the interaction with Mif2, little is known about how COMA is recruited to the centromeric nucleosome and how this may be regulated during the cell cycle. Ctf19 was shown to interact by two-hybrid analysis with Cse4 (Ortiz et al, 1999), but this has not been confirmed using biochemical methods. Furthermore, Okp1/Ame1 interacts with DNA, but the interaction is not specific to centromeric sequences (Hornung et al, 2014). ...
... Several mutants were isolated, and the site of the extragenic suppressor was determined by whole-genome sequencing. Interestingly, we obtained one isolate that carried a mutation in OKP1, which encodes the COMA component Okp1 (Ortiz et al, 1999). The mutation causes the change of arginine 164 of Okp1 to cysteine (okp1-R164C), a residue that lies within the "core" region (amino acids 166-211) that is essential for viability (Schmitzberger et al, 2017). ...
Kinetochores are supramolecular assemblies that link centromeres to microtubules for sister chromatid segregation in mitosis. For this, the inner kinetochore CCAN/Ctf19 complex binds to centromeric chromatin containing the histone variant CENP-A, but whether the interaction of kinetochore components to centromeric nucleosomes is regulated by posttranslational modifications is unknown. Here, we investigated how methylation of arginine 37 (R37Me) and acetylation of lysine 49 (K49Ac) on the CENP-A homolog Cse4 from Saccharomyces cerevisiae regulate molecular interactions at the inner kinetochore. Importantly, we found that the Cse4 N-terminus binds with high affinity to the Ctf19 complex subassembly Okp1/Ame1 (CENP-Q/CENP-U in higher eukaryotes), and that this interaction is inhibited by R37Me and K49Ac modification on Cse4. In vivo defects in cse4-R37A were suppressed by mutations in OKP1 and AME1, and biochemical analysis of a mutant version of Okp1 showed increased affinity for Cse4. Altogether, our results demonstrate that the Okp1/Ame1 heterodimer is a reader module for posttranslational modifications on Cse4, thereby targeting the yeast CCAN complex to centromeric chromatin.
... A multiple sequence alignment (MSA) of Cse4 CENP-A protein sequences ( Figure 2A) unveiled a conserved region (ScCse4 aa 34-61) unique to Cse4 proteins of inter-related yeasts which is almost identical to the essential N-terminal Domain (END, aa 28-60). The END domain was previously shown to be required for Cse4 function and for recruiting the 'Mcm21p/Ctf19p/Okp1p complex' to minichromosomes (Chen et al., 2000, Ortiz et al., 1999. ...
... The COMA complex is composed of two essential, Ame1/Okp1, and two non-essential, Ctf19/Mcm21, subunits (Ortiz et al., 1999, Cheeseman et al., 2002. Both, Ctf19 and Mcm21 contain C-terminal tandem-RWD (RING finger and WD repeat containing proteins and DEADlike helicases) domains forming a rigid heterodimeric Y-shaped scaffold whose respective Nterminal RWDs of the tandems pack together as revealed by a recent crystal structure of the K. lactis complex (Schmitzberger and Harrison, 2012). ...
... Furthermore, deletion of the N-terminal 50 amino acids of Cse4 was found to be lethal (Keith et al., 1999), whereas the H3 N-terminus was dispensable for viability (Mann and Grunstein, 1992), and END was previously implicated in interacting with the 'Mcm21/Okp1/Ctf19' complex (Ortiz et al., 1999, Chen et al., 2000. Consistent with these previous findings, we narrowed down the essential N-terminal Cse4 patch to amino acids 34-46 which was identified as the minimal motif sufficient for the interaction with Ame1/Okp1. ...
Kinetochores are macromolecular protein complexes assembled on centromeric chromatin that ensure accurate chromosome segregation by linking DNA to spindle microtubules and integrating safeguard mechanisms. A kinetochore-associated pool of Ipl1Aurora B kinase, a subunit of the chromosomal passenger complex (CPC), was previously implicated in feedback control mechanisms. To study the kinetochore subunit connectivity built on budding yeast point centromeres and its CPC interactions we performed crosslink-guided in vitro reconstitution. The Ame1/Okp1CENP-U/Q heterodimer, forming the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus and thereby establishes a direct link to the outer kinetochore MTW1 complex. The Sli15/Ipl1INCENP/Aurora B core-CPC interacted with COMA through the Ctf19 C-terminus, and artificial tethering of Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19/Mcm21 deletion in a Sli15 centromere-targeting deficient mutant. This study reveals characteristics of the inner kinetochore architecture assembled at point centromeres and the relevance of its Sli15/Ipl1 interaction for CPC function.
... There is increasing genetic and biochemical evidence for a regulated network of protein-protein and protein-DNA interactions that contributes to the structure of the S.cerevisiae kinetochore complex (23). The CBF3 multiprotein complex consists of the four essential subunits: p110, p64, p58 and p23, which form the core element of the centromere (reviewed in 5). ...
... We are not aware of reports on other biological systems with such a DNA sequence and binding strength variation but common function within one cell. It is likely that a variety of protein-protein and protein-DNA interactions are necessary for the formation of fully assembled centromere complexes (23). Thus, the binding strength variations observed in our system represent only one contributing part of the biologically active complex. ...
... It is possible that similar mechanisms act at centromeres. There are more than 12 established kinetochore proteins contributing to structure and function of the centromeres in budding yeast (23). Based on biochemical and genetic data, the centromere proteins Mif2p, Cse4p, Mcm21p and the Cbf3 subunits p110 and p64 have been proposed as Cbf1p-interacting proteins (18,23,24,33). ...
Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix–loop–helix leucine zipper (bHLHzip) family of DNA binding proteins.
Cbf1p binds to a conserved element in the 5′-flanking region of methionine biosynthetic genes and to centromere DNA element
I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs
in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants
of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in
the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions
on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest
that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute
to the complex formation between Cbf1p and CEN DNA.
... Whereas CDEIII and CBF3 are essential for viability (Doheny et al., 1993;Goh and Kilmartin, 1993;Hegemann et al., 1988;Jehn et al., 1991;Lechner and Carbon, 1991;McGrew et al., 1986;Ng and Carbon, 1987;Panzeri et al., 1985), cells with CDEI disrupted remain viable but exhibit mitotic chromosome loss, and defective centromere function in meiosis I (Cumberledge and Carbon, 1987;Gaudet and Fitzgerald-Hayes, 1989;Hegemann et al., 1988;Niedenthal et al., 1991;Panzeri et al., 1985). CDEII is less well conserved, however, its AT-rich DNA sequence is proposed to be favorable for CENP-A Nuc wrapping due to its increased tendency to curve (Bechert et al., 1999;Koo et al., 1986;Murphy et al., 1991;Ortiz et al., 1999). Indeed, CDEII is necessary for optimal centromere function: reduction of AT-content, disruption of polyA/T tracts and alteration to CDEII length, result in mitotic delay and chromosome segregation defects (Baker and Rogers, 2005;Cumberledge and Carbon, 1987;Fitzgerald-Hayes, 1987, 1989;Murphy et al., 1991;Spencer and Hieter, 1992). ...
... However, since CBF3 remains localized at the centromere during metaphase and anaphase (Cieslinski et al., 2021), it likely has a structural role in kinetochore assembly beyond CENP-A Nuc deposition. The findings described above paint a molecular picture of the yeast inner kinetochore consisting of CCAN, CBF1 and CBF3 assembled onto CENP-A Nuc :CENP-C (Akiyoshi et al., 2009;Cheeseman et al., 2002;De Wulf et al., 2003;Ortiz et al., 1999). We previously determined a cryo-EM structure of S. cerevisiae CCAN in complex with CENP-A Nuc reconstituted with non-native Widom 601 DNA (Yan et al., 2019). ...
The point centromere of budding yeast specifies assembly of the large multi-subunit kinetochore complex. By direct attachment to the mitotic spindle, kinetochores couple the forces of microtubule dynamics to power chromatid segregation at mitosis. Kinetochores share a conserved architecture comprising the centromere-associated inner kinetochore CCAN (constitutive centromere-associated network) complex and the microtubule-binding outer kinetochore KMN network. The budding yeast inner kinetochore additionally includes the centromere-binding CBF1 and CBF3 complexes. Here, we reconstituted the complete yeast inner kinetochore complex assembled onto the centromere-specific CENP-A nucleosome (CENP-ANuc) and determined its structure using cryo-EM. This revealed a central CENP-ANuc, wrapped by only one turn of DNA, and harboring extensively unwrapped DNA ends. These free DNA duplexes function as binding sites for two CCAN protomers, one of which entraps DNA topologically and is positioned precisely on the centromere by the sequence-specific DNA-binding complex CBF1. The CCAN protomers are connected through CBF3 to form an arch-like configuration, binding 150 bp of DNA. We also define a structural model for a CENP-ANuc-pathway to the outer kinetochore involving only CENP-QU. This study presents a framework for understanding the basis of complete inner kinetochore assembly onto a point centromere, and how it organizes the outer kinetochore for robust chromosome attachment to the mitotic spindle.
... Because the Ctf19c CCAN persists on centromeres through all stages of vegetative growth and gametogenesis, we hypothesised that it plays a critical role in this re-structuring. Ctf19c CCAN proteins that are dispensable for vegetative growth (Measday et al., 2002;Meluh and Koshland, 1997;Ortiz et al., 1999;Pot et al., 2003;De Wulf et al., 2003) become essential for chromosome segregation during meiosis (Fernius and Marston, 2009; and Ctf19 CENP-P is implicated in meiotic kinetochore assembly (Mehta et al., 2014). Consistently, cells lacking the non-essential Ctf19 CENP-P , Mcm21 CENP-O , Chl4 CENP-N or Iml3 CENP-L show ~50% wild-type viability after mitosis ( Figure 3A), while only around ~1-10% of tetrads produced four viable spores after meiosis ( Figure 3B). ...
... https://doi.org/10.1101 ndt80Δ (Vincenten et al., 2015), CEN5-GFP dots (Toth et al., 2000), okp1-5 (Ortiz et al., 1999), PDS1-tdTomato and HTB1-mCherry (Matos et al., 2008), pCUP1-IME1/pCUP1-IME4 (Chia and van Werven, 2016) and ndc80(∆2-28) were described previously. ...
Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganisation of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that replace the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that this kinetochore remodelling in meiosis requires the Ctf19c CCAN inner kinetochore complex. Through functional analyses, we identify a Ctf19c CCAN -dependent kinetochore assembly pathway that is dispensable for mitotic growth, but becomes critical upon meiotic entry. Therefore, extensive kinetochore remodelling and a distinct assembly pathway direct the specialization of meiotic kinetochores for successful gametogenesis.
... As discussed above, CENP-C and CENP-N have both been shown to directly interact with CENP-A and serve as important connections between the inner kinetochore and centromeric chromatin. Most human CCAN proteins have orthologues in the budding yeast (S. cerevisiae) Ctf19 complex, which also contains the CBF3 complex that directly binds yeast centromeric DNA and is not present in humans [25,37,[140][141][142][143]. ...
... Budding yeast do not have a homologue for CENP-R, but the yeast COMA complex is equivalent to human CENP-OPQU (figure 5d). The COMA complex is made up of CENP-P Ctf19 , CENP-Q Okp1 , CENP-O Mcm21 and CENP-U Ame1 [37,141]. CENP-U Ame1 has been shown to interact with the outer kinetochore MIND complex, analogous to the human Mis12 complex [150], discussed below. ...
Eukaryotic chromosome segregation relies upon specific connections from DNA to the microtubule-based spindle that forms at cell division. The chromosomal locus that directs this process is the centromere, where a structure called the kinetochore forms upon entry into mitosis. Recent crystallography and single-particle electron microscopy have provided unprecedented high-resolution views of the molecular complexes involved in this process. The centromere is epigenetically specified by nucleosomes harbouring a histone H3 variant, CENP-A, and we review recent progress on how it differentiates centromeric chromatin from the rest of the chromosome, the biochemical pathway that mediates its assembly and how two non-histone components of the centromere specifically recognize CENP-A nucleosomes. The core centromeric nucleosome complex (CCNC) is required to recruit a 16-subunit complex termed the constitutive centromere associated network (CCAN), and we highlight recent structures reported of the budding yeast CCAN. Finally, the structures of multiple modular sub-complexes of the kinetochore have been solved at near-atomic resolution, providing insight into how connections are made to the CCAN on one end and to the spindle microtubules on the other. One can now build molecular models from the DNA through to the physical connections to microtubules.
... To address whether the observed kinetochore declustering is specific to the loss of Dam1 positioned at the kinetochoremicrotubule interface, we investigated the kinetochore clustering in the absence of Ctf19, a protein residing at the middle of the kinetochore away from the interface (Cheeseman et al. 2002;Ortiz et al. 1999). Live cell analysis confirmed the previous reports where clustering defect is not observed in the absence of Ctf19 with or without the microtubules (Fig. S2, Mehta et al. 2014). ...
... Dam1 plays a role in stabilization of Cse4 at the centromere Since in the absence of Dam1 we observed a reduced level of Ndc10 at the centromere and as Ndc10 acts upstream of the Cse4 deposition at the centromere (Camahort et al. 2007;Ortiz et al. 1999), the enrichment of Cse4 was verified in Dam1-depleted condition. Live cell imaging using internally tagged Cse4-GFP (Kosco et al. 2001) showed that in contrast to Ndc10, Cse4-GFP signal was completely absent in 72% of Dam1-depleted cells as compared to the wild type, where the signal appeared as tight-knit clustered foci in~95% of the cells (Fig. 2a). ...
A higher order organization of the centromeres in the form of clustering of these DNA loci has been observed in many organisms. While centromere clustering is biologically significant to achieve faithful chromosome segregation, the underlying molecular mechanism is yet to be fully understood. In budding yeast, a kinetochore-associated protein Slk19 is shown to have a role in clustering in association with the microtubules whereas removal of either Slk19 or microtubules alone does not have any effect on the centromere clustering. Furthermore, Slk19 is non-essential for growth and becomes cleaved during anaphase whereas clustering being an essential event occurs throughout the cell cycle. Hence, we searched for an additional factor involved in the clustering and since the integrity of the kinetochore complex is shown to be crucial for centromere clustering, we restricted our search within the complex. We observed that the outermost kinetochore protein Dam1 promotes centromere clustering through stabilization of the kinetochore integrity. While in the absence of Dam1 we failed to detect Slk19 at the centromere, on the other hand, we found almost no Dam1 at the centromere in the absence of Slk19 and microtubules suggesting interdependency between these two pathways. Strikingly, we observed that overexpression of Dam1 or Slk19 could restore the centromere clustering largely in the cells devoid of Slk19 and microtubules or Dam1, respectively. Thus, we propose that in budding yeast, centromere clustering is achieved at least by two parallel pathways, through Dam1 and another via Slk19, in concert with the microtubules suggesting that having a dual mechanism may be crucial for ensuring microtubule capture by the point centromeres where each attaches to only one microtubule.
... The evolutionarily conserved C-terminus histone fold domain (HFD) carries a centromere targeting domain, which is essential for recruitment and incorporation of Cse4 into the CEN chromatin (Meluh et al., 1998;Keith et al., 1999). The N-terminus of Cse4 (∼129 amino acids) interacts with kinetochore proteins such as the components of the COMA complex (Ctf19, Okp1, Mcm21, and Ame1) and facilitates their recruitment to the CEN (Ortiz et al., 1999). Moreover, the N-terminus of CENP-A also directs the targeting of other kinetochore proteins to the CEN (Van Hooser et al., 2001). ...
... With the exception of one serine, eight of the nine Cse4 serine residues that are phosphorylated by Cdc5 are in the N-terminus of Cse4 ( Figure 3A). Cse4 interacts in vivo with Ctf19 and Mcm21 (Ortiz et al., 1999;Ranjitkar et al., 2010), and this interaction is mediated by the N-terminus of Cse4 . Genetic interactions have also been reported for mutants of cse4 with ctf19Δ and mcm21Δ (Samel et al., 2012). ...
Evolutionarily conserved polo-like kinase, Cdc5 (Plk1 in humans), associates with kinetochores during mitosis; however, the role of cell cycle-dependent centromeric ( CEN) association of Cdc5 and its substrates that exclusively localize to the kinetochore have not been characterized. Here we report that evolutionarily conserved CEN histone H3 variant, Cse4 (CENP-A in humans), is a substrate of Cdc5, and that the cell cycle-regulated association of Cse4 with Cdc5 is required for cell growth. Cdc5 contributes to Cse4 phosphorylation in vivo and interacts with Cse4 in mitotic cells. Mass spectrometry analysis of in vitro kinase assays showed that Cdc5 phosphorylates nine serine residues clustered within the N-terminus of Cse4. Strains with cse4-9SA exhibit increased errors in chromosome segregation, reduced levels of CEN-associated Mif2 and Mcd1/Scc1 when combined with a deletion of MCM21. Moreover, the loss of Cdc5 from the CEN chromatin contributes to defects in kinetochore integrity and reduction in CEN-associated Cse4. The cell cycle-regulated association of Cdc5 with Cse4 is essential for cell viability as constitutive association of Cdc5 with Cse4 at the kinetochore leads to growth defects. In summary, our results have defined a role for Cdc5-mediated Cse4 phosphorylation in faithful chromosome segregation.
... CENP-CHIKMLN is also required for recruitment of a 5-subunit complex incorporating the CENP-O, CENP-P, CENP-Q, CENP-U, and CENP-R subunits (CENP-OPQUR, whose subunits are schematically shown in Figure 1B) (Eskat et al., 2012;Foltz et al., 2006;Hori et al., 2008b;McKinley et al., 2015;Minoshima et al., 2005;Okada et al., 2006;Samejima et al., 2015). CENP-OPQUR is related to the COMA com- plex of S. cerevisiae (De Wulf et al., 2003;Hori et al., 2008b;Hyland et al., 1999;Ortiz et al., 1999;Schmitzberger et al., 2017;Westermann et al., 2003). Its precise role at kinetochores remains poorly characterized, but it consists at least in part in the recruitment of other kinetochore residents, including the microtubule plus-end directed motor CENP-E and Polo-like kinase 1 (Plk1), the latter through phosphorylation of CENP-U ( Bancroft et al., 2015;Hori et al., 2008b;Kang et al., 2006). ...
... Human CENP-OPQUR did not bind to the NDC80C or to the entire KMN ( Figures S4C and S4D), nor did it bind to complexes of MIS12C or MIS12C:NDC80C with CENP-C 1-544 ( Figures S4E and S4F), a crucial link between the inner and outer kinetochore ( Dimitrova et al., 2016;Gascoigne et al., 2011;Petrovic et al., 2016;Przewloka et al., 2011;Screpanti et al., 2011;Weir et al., 2016). Thus, CENP-OPQUR does not contribute to the stabiliza- tion of the connection between the inner and the outer kineto- chore, a function that has instead been described for its ortholog in S. cerevisiae (the COMA complex, comprising the Ctf19 CENP-P , Okp1 CENP-Q , Mcm21 CENP-O , and Ame1 CENP-U subunits and lack- ing a clear CENP-R ortholog) (De Wulf et al., 2003;Dimitrova et al., 2016;Hornung et al., 2014;Hyland et al., 1999;Ortiz et al., 1999;Pekg? z Altunkaya et al., 2016;Schmitzberger and Harrison, 2012;Westermann et al., 2003). ...
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.
... COMA connects centromereassociated proteins and outer kinetochore (Hornung et al, 2014;Dimitrova et al, 2016). COMA includes the proteins "associated with microtubules and essential 1" (Ame1) (Cheeseman et al, 2002), "chromosome transmission fidelity 19" (Ctf19) (Hyland et al, 1999), "minichromosome maintenance 21" (Mcm21) (Poddar et al, 1999) and "outer kinetochore protein 1" (Okp1) (Ortiz et al, 1999). These proteins are part of a supramolecular assembly from the inner kinetochore, termed CTF19, which contains eight other subunits that include "non-essential kinetochore protein 1" (Nkp1) and "nonessential kinetochore protein 2" (Nkp2) (Cheeseman et al, 2002;De Wulf et al, 2003;Schleiffer et al, 2012). ...
... The specific functional relevance of several CCAN subunits is unclear. Ame1 CENP-U and Okp1 CENP-Q (superscripts are human orthologue names; or in the following, in the case of human protein names, budding yeast-orthologue names) are essential for viability (essential) of S. cerevisiae (Ortiz et al, 1999;Cheeseman et al, 2002;Hornung et al, 2014). ...
Kinetochores are dynamic cellular structures that connect chromosomes to microtubules. They form from multi-protein assemblies that are evolutionarily conserved between yeasts and humans. One of these assemblies-COMA-consists of subunits Ame1(CENP-U), Ctf19(CENP-P), Mcm21(CENP-O) and Okp1(CENP-Q) A description of COMA molecular organization has so far been missing. We defined the subunit topology of COMA, bound with inner kinetochore proteins Nkp1 and Nkp2, from the yeast Kluyveromyces lactis, with nanoflow electrospray ionization mass spectrometry, and mapped intermolecular contacts with hydrogen-deuterium exchange coupled to mass spectrometry. Our data suggest that the essential Okp1 subunit is a multi-segmented nexus with distinct binding sites for Ame1, Nkp1-Nkp2 and Ctf19-Mcm21. Our crystal structure of the Ctf19-Mcm21 RWD domains bound with Okp1 shows the molecular contacts of this important inner kinetochore joint. The Ctf19-Mcm21 binding motif in Okp1 configures a branch of mitotic inner kinetochores, by tethering Ctf19-Mcm21 and Chl4(CENP-N)-Iml3(CENP-L) Absence of this motif results in dependence on the mitotic checkpoint for viability.
... Cohesin is an essential protein complex that facilitates spindle attachment to the centromere. Mcm21 is a non-essential kinetochore component of the COMA complex [14] that is responsible for the enrichment of cohesin at the pericentromeric region. Deletion of MCM21 results in a partial dispersal of kinetochores from the normal cluster around the SPB, but does not prevent relatively normal chromosome segregation [15]. ...
... Despite reports that deletion of MCM21 leads to the partial dislocation of centromeres from the SPB [14], this deletion did not relieve the constraint of centromere proximity in DSB repair. However, disruption of CEN2 function by galactose-induced transcription proved to cause a modest but statistically significant increase in the ability of centromere-proximal sequences to recombine. ...
Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238 kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor. © 2017 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalauthor and source are credited.
... Thus, more likely, the direct interactor of Cse4 is a component of the inner kinetochore. A link between the Cse4 Nterminus and Ctf19, the naming component of the Ctf19 complex, has been shown by two-hybrid analysis (Ortiz et al. 1999), and this interaction therefore may be modulated by PTMs on Cse4. Alternatively, the interactor may be another COMA component. ...
... Okp1 and Ame1 are parts of the COMA complex and are essential for a stable checkpoint arrest in Saccharomyces cerevisiae (De Wulf, McAinsh and Sorger 2003;Pot et al. 2005). Previous work showed that okp1-5 cells have a strong G2/M arrest (Ortiz et al. 1999), whereas ame1-4 cells do not maintain the arrest (Pot et al. 2005). Thus, ubr2 may be able to suppress a weak, but not a strong, cell cycle defect. ...
The kinetochore, a supramolecular protein complex, provides the physical connection between chromatin and the microtubule and ensures correct chromosome segregation during mitosis. Centromeric regions are marked by the presence of the histone H3 variant CENP-A. Cse4, the CENP-A homolog from Saccharomyces cerevisiae, is methylated on arginine 37 in its N-terminus (R37), and the absence of methylation (cse4-R37A) causes synthetic genetic defects in combination with mutations or deletions in genes encoding components of the Ctf19/ CCAN complex and with the CDEI binding protein Cbf1. Here, we report that the absence of the E3 ubiquitin ligase Ubr2 as well as its adaptor protein Mub1 suppresses the defects caused by the absence of Cse4-R37 methylation. Ubr2 is known to regulate the levels of the MIND complex component Dsn1 via ubiquitination and proteasome-mediated degradation. Accordingly, we found that overexpression of DSN1 also led to suppression of Cse4 methylation defects. Altogether, our data indicate that the absence of R37 methylation reduces the recruitment of kinetochore proteins to centromeric chromatin, and that this can be compensated for by stabilizing the outer kinetochore protein Dsn1.
... All other components are interdependent on each other for centromere localization and require the CENP-H-K-I complex (Okada et al. 2006, Hori et al. 2008b. The homologous complex, called COMA, had been isolated earlier from budding yeast, where the CENP-U/Ame1 and CENP-Q/Okp1 subunits are essential (Ortiz et al. 1999). CENP-U mutants are viable in certain mammalian cell lines but essential in mouse embryos and embryonic stem cells (Kagawa et al. 2014). ...
... Thus Ame1 to Mis12 complex interactions are critical for inner-outer kinetochore linkage in budding yeast. Deletion of the Ame1 N-terminal motif reduced both Mis12 and COMA complex localization, suggesting interdependence for assembly, but because Ame1-Okp1 can bind directly to CENP-C/Mif2 and to DNA, at least in vitro, and it was suggested that it forms a link between the CDEIII-binding CBF3 complex and the KMN (Ortiz et al. 1999), it remains possible that there are secondary effects. These interactions have not been studied in any great detail, however. ...
Chromosome segregation relies on coordinated activity of a large assembly of proteins, the kinetochore interaction network (KIN). How conserved the underlying mechanisms driving the epigenetic phenomenon of centromere and kinetochore assembly and maintenance are remains unclear, even though various eukaryotic models have been studied. More than 50 different proteins, many in multiple copies, comprise the KIN or are associated with fungal centromeres and kinetochores. Proteins isolated from immune sera recognized centromeric regions on chromosomes and thus were named centromere proteins (CENPs). CENP-A, sometimes called centromere-specific H3 (CenH3), is incorporated into nucleosomes within or near centromeres. The constitutive centromere-associated network (CCAN) assembles on this specialized chromatin, likely based on specific interactions with and requiring presence of CENP-C. The outer kinetochore comprises the Knl1-Mis12-Ndc80 (KMN) protein complexes that connect CCAN to spindles, accomplished by binding and stabilizing microtubules (MTs) and in the process generating load-bearing assemblies for chromatid segregation. In most fungi the Dam1/DASH complex connects the KMN complexes to MTs. Fungi present a rich resource to investigate mechanistic commonalities but also differences in kinetochore architecture. While ascomycetes have sets of CCAN and KMN proteins that are conserved with those of budding yeast or metazoans, searching other major branches of the fungal kingdom revealed that CCAN proteins are poorly conserved at the primary sequence level. Several conserved binding motifs or domains within KMN complexes have been described recently, and these features of ascomycete KIN proteins are shared with most metazoan proteins. In addition, several ascomycete-specific domains have been identified here.
... In particular, COMA function has been described in a hierarchical cascade as a recruitment factor of other linker components, such as MIND (Mtw1, Nnf1, Nsl1, Dsn1), Ctf3 and Chl4/Iml3. MIND as well as COMA mutants show unstable spindle and monopolar attachment phenotypes, and consistent with the activation of the spindle checkpoint, cells show a mitotic delay (De Wulf et al., 2003;Foley and Kapoor, 2013;Ortiz et al., 1999;Scharfenberger et al., 2003;Stoler et al., 1995). ...
... It was shown before that the subunits of the COMA complex Ctf19, Okp1 and Mcm21, are components of the linker layer of the yeast kinetochore. Further it was shown that this complex links CBF3, which directly binds the CDEIII element of the centromeric DNA, to further components of the centromere, Cbf1 and Cse4 (Ortiz et al., 1999). Whereas direct biochemical interaction of Cse4 and the COMA components was detected in a Yeast-2-hybrid assay, in this study, we could identify Ctf19 as an interaction partner of Cse4 via immunoprecipitation, and that the interaction is dependent on the acetylation of K49 in the N-terminus of Cse4. ...
Die zentromerischen Regionen eukaryotischer Chromosomen sind notwendig für die Segregation der Schwesternchromatiden während der Zellteilung. In den Nukleosomen in diesen Regionen ist das kanonische Histon H3 durch die zentromerische Histon H3 Variante CENP-A ersetzt. Diese wird in Saccharomyces cerevisiae Cse4 genannt. Indem CENP-A die geordnete Assemblierung einzelner Untereinheiten des Kinetochors vermittelt, spezifiziert es die zentromerische Chromosomenregion und ist damit essentiell für die korrekte Segregation der Chromosomen während der Zellteilung. Kürzlich wurde gezeigt, dass Cse4 posttranslational modifiziert wird. Von Bedeutung für diese Arbeit sind die in der essentiellen Domäne des N-terminus liegenden Methylierung von Arginin 37 und die Acetylierung von Lysin 49, die durch phänotypische Suppression eine genetische Interaktion und eine antagonistische Funktion bei der epigenetischen Regulation in der korrekten Assemblierung der Kinetochoruntereinheiten zeigen. In dieser Arbeit wurde gezeigt, dass die Acetylierung an Cse4K49 von der Histonacetyltransferase Gcn5 abhängt, und dass dieses Enzym Komponenten des SAGA-Komplexes, aber nicht des ADA-Komplexes benötigt. Außerdem konnte in dieser Arbeit gezeigt werden, dass die Acetylierung von K49 in der frühen S-Phase ansteigt und zum Ende der S-Phase abnimmt. Es konnte weiterhin eine biochemische Interaktion der N-terminalen Domäne von Cse4 mit der COMA-Untereinheit Ctf19, der zentralen Region des Kinetochors, nachgewiesen werden, möglicherweise mit einer Präferenz zu einer nicht acetylierten Form von Cse4K49. In dieser Arbeit konnte gezeigt werden, dass der Transkriptions-Co-Aktivator Komplex SAGA eine Funktion an der zentromerischen Region in S. cerevisiae aufweist. Des Weiteren ist die Acetylierung an Cse4K49 in die Rekrutierung der Kinetochoruntereinheit Ctf19 involviert, und gibt damit einen Hinweis auf einen epigenetischen Regulationsmechanismus während der Chromosomensegregation.
... In addition to its inner kinetochore localization, Ndc10 has been also shown locate to the mitotic spindle (Bouck and Bloom 2005). CBF3-complex dependency has been shown for all known kinetochore proteins, including Cse4 (Ortiz, Stemmann et al. 1999). ...
... Ever since the three proteins, Ctf19, Mcm21 and Okp1, have been identified by onehybrid screen, and confirmed by the ChIP analysis to be constituents of the budding yeast kinetochore, it was speculated that they form a complex. It was proposed that this protein complex most likely links CBF3 with further kinetochore components of the budding yeast centromere; Mif2, Cse4 and Cbf1 proteins (Ortiz, Stemmann et al. 1999). ...
The kinetochore is a specialized structure composed of centromeric DNA and a large number of proteins. The primary function of the kinetochore is to connect chromosomes with the mitotic spindle throughout the cell cycle and to monitor the fidelity of these attachments in order to ensure proper chromosome segregation. Despite the fact that chromosome segregation is directed by the kinetochore, the architecture and assembly of such an intricate structure remains elusive. We use budding yeast as a model system to characterize direct interactions among the central domain of the kinetochore proteins, specifically the COMA-complex. The COMA-complex consists of four proteins; two nonessential proteins Ctf19 and Mcm21 and two essential proteins Ame1 and Okp1. Although the chromosome segregation is a highly conserved process, the human orthologues of the two essential Ame1 and Okp1 proteins have not been identified. The tetrameric complex is the core of the COMA-network which is composed of seven additional nonessential proteins: Ctf3, Mcm16, Mcm22, Chl4, Iml3, Nkp1 and Nkp2, more loosely associated. According to the central localization within the kinetochore, the COMA-complex represents one of the linker complexes (together with the Mtw1-, Ndc80- and Spc105-complexes) bridging the centromere-associate inner proteins with microtubule-bounded outer kinetochore proteins. Here we present a biochemical approach to reconstitute and to characterize the budding yeast COMA-network. Our first aim was to reconstitute the COMA-complex in vitro and in vivo. A stabile heterodimer consisting of Ctf19 and Mcm21 proteins could be reconstituted as a tetrameric complex in solution. The Ame1 and Okp1 heterodimer showed noticeable instability and we were not able to reconstitute it. Surprisingly, the trimeric Ctf19, Mcm21 and Okp1-complex could be assembled in vivo independently of the Ame1 essential protein. Moreover, we demonstrated that the Okp1 coiled-coil region per se is sufficient to form a complex with Ctf19 and Mcm21 proteins in vitro. The tetrameric COMA-complex could have also been reconstituted in vitro, but the amount and the stoichiometry of the components were not satisfactory. In solution, the COMA-complex showed oligomerization behavior. Through the protein purification experiments, we also found that the Ctf19, Mcm21 and Okp1-complex as well as the Ame1 protein separately may bind to unspecific, E.coli RNA. To determine other kinetochore subunits that can directly or indirectly associate with the COMA-components, we performed co-immunoprecipitation from yeast cells with either Okp1-TAP or Ame1-TAP tagged proteins. Among many known interacting partners, the Dsn1 component of the Mtw1-central kinetochore complex was identified. To support this finding and to test if the binding between the Dsn1 and the COMA-proteins is direct or indirect, we performed in vitro reticulocyte lysate binding assay. The interaction between the Mtw1- and the COMA-complexes via the Dsn1 protein was confirmed. Additional information has been gained from the co-immunoprecipitation experiments using budding yeast cells. We identified two proteins from the COMA-network, Nkp1 and Nkp2 proteins, as highly enriched. Since this may reflect the close proximity of these two proteins to the core of the COMA-network, we purified separately Nkp1 and Nkp2 dimer (which revealed the stabile heterodimer formation between these two Nkp proteins), combined it with the recombinant COMA-complex and reconstituted the hexameric protein complex at a 1:1:1:1:1:1 stoichiometry. Taken together, this study led to proposal of a new model for the spatial organization of the COMA-network. In summary, we used affinity based protein isolation to identify new direct binding partners within the central domain of the budding yeast kinetochore. Our findings improve the current understanding of the overall kinetochore architecture. The complete characterization of the kinetochore structure and organization has to be fully known, ultimately leading to three-dimensional vision and biochemical features of the kinetochore complexes, in order to unravel the mechanisms of chromosome segregation and maintenance of genome stability. Our work is therefore one step further in answering relevant biological and medical questions concerning faithful chromosome segregation during mitosis.
... Whereas CDEIII and CBF3 are essential for viability (19)(20)(21)(22)(23)(24)(25)(26), cells with CDEI disrupted remain viable but exhibit mitotic chromosome loss, and in meiosis I, defective centromere function (20,23,(27)(28)(29). CDEII is less well conserved; however, its AT-rich DNA sequence is proposed to be favorable for CENP-A Nuc assembly in vivo because of its increased tendency to curve (30)(31)(32)(33). ...
The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
... Rapamycin addition leads to the stable interaction of FRB and FKBP12, which results in the removal of Ctf19 from the nucleus along with the ribosomal subunit (Haruki et al., 2008). Although Ctf19 is a nonessential kinetochore component in mitosis, the kinetochore is extensively reorganized during meiosis and Ctf19 has a crucial role in kinetochore reassembly after cells exit prophase I (Hyland et al., 1999;Ortiz et al., 1999;Borek et al., 2021). To avoid altering kinetochore structure, we depleted Ctf19 from the nucleus after kinetochore reassembly. ...
Proper chromosome segregation depends on establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests a model that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.
... Rapamycin addition leads to the stable interaction of FRB and FKBP12, which results in the removal of Ctf19 from the nucleus along with the ribosomal subunit (Haruki et al., 2008). Although Ctf19 is a non-essential kinetochore component in mitosis, the kinetochore is extensively reorganized during meiosis and Ctf19 has a crucial role in kinetochore re-assembly after cells exit prophase I (Borek et al., 2021;Hyland et al., 1999;Ortiz et al., 1999). To avoid altering kinetochore structure, we depleted Ctf19 from the nucleus after kinetochore re-assembly. ...
Proper chromosome segregation depends on establishment of bioriented kinetochore-microtubule attachments, which often requires multiple rounds of release and reattachment. Aurora B and C kinases phosphorylate kinetochore proteins to release tensionless attachments. Multiple pathways recruit Aurora B/C to the centromere and kinetochore. We studied how these pathways contribute to anaphase onset timing and correction of kinetochore-microtubule attachments in budding yeast meiosis and mitosis. We find that the pool localized by the Bub1/Bub3 pathway sets the normal duration of meiosis and mitosis, in differing ways. Our meiosis data suggests that disruption of this pathway leads to PP1 kinetochore localization, which dephosphorylates Cdc20 for premature anaphase onset. For error correction, the Bub1/Bub3 and COMA pathways are individually important in meiosis but compensatory in mitosis. Finally, we find that the haspin and Bub1/3 pathways function together to ensure error correction in mouse oogenesis. Our results suggest that each recruitment pathway localizes spatially distinct kinetochore-localized Aurora B/C pools that function differently between meiosis and mitosis.
... 3.2.1 Impact of CCAN subunit disruption-In budding yeast, three CCAN subunits (Okp1/Ame1/Mif2) are encoded on essential genes while the remainder are indispensable for viability -albeit associated with increased frequency of chromosome mis-segregation (54,68,164,185,200). The picture in humans is complicated since: (1) results from acute or chronic knockdown/out experiments can vary with regard to the penetrance of chromosome alignment phenotypes; and (2) there is emerging evidence of cell type specific requirements (72,163,177,192,202). Differences in essentiality may also underlie the different extent to which different organisms rely on functional modules linking the centromeric nucleosome to the microtubule i.e. there are a few different molecular paths which involve different interactions between the CenpC and KMN which are not as used in some organisms compared to others (see below for details). ...
Kinetochores are molecular machines that power chromosome segregation during the mitotic and meiotic cell divisions of all eukaryotes. Aristotle explains how we think we have knowledge of a thing only when we have grasped its cause. In our case, to gain understanding of the kinetochore, the four causes correspond to questions that we must ask: ( a) What are the constituent parts, ( b) how does it assemble, ( c) what is the structure and arrangement, and ( d) what is the function? Here we outline the current blueprint for the assembly of a kinetochore, how functions are mapped onto this architecture, and how this is shaped by the underlying pericentromeric chromatin. The view of the kinetochore that we present is possible because an almost complete parts list of the kinetochore is now available alongside recent advances using in vitro reconstitution, structural biology, and genomics. In many organisms, each kinetochore binds to multiple microtubules, and we propose a model for how this ensemble-level architecture is organized, drawing on key insights from the simple one microtubule–one kinetochore setup in budding yeast and innovations that enable meiotic chromosome segregation.
Expected final online publication date for the Annual Review of Genetics, Volume 56 is November 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Yet, CENP-C Mif2 , CENP-Q Okp1 , and CENP-U Ame1 are the only CCAN Ctf19C components that are essential for viability (Biggins, 2013). CENP-Q Okp1 and CENP-U Ame1 form a dimeric subcomplex which along with CENP-O Mcm21 and CENP-P Ctf19 constitute the COMA complex (Hyland et al., 1999;Ortiz et al., 1999;Poddar et al., 1999;Cheeseman et al., 2002). CENP-Q Okp1 and CENP-U Ame1 contain a coiled-coil region in the C-terminal half and harbor no other distinct structural domains ( Figure 2C) (Hornung et al., 2014). ...
The assembly of a functional kinetochore on centromeric chromatin is necessary to connect chromosomes to the mitotic spindle, ensuring accurate chromosome segregation. This connecting function of the kinetochore presents multiple internal and external structural challenges. A microtubule interacting outer kinetochore and centromeric chromatin interacting inner kinetochore effectively confront forces from the external spindle and centromere, respectively. While internally, special inner kinetochore proteins, defined as “linkers,” simultaneously interact with centromeric chromatin and the outer kinetochore to enable association with the mitotic spindle. With the ability to simultaneously interact with outer kinetochore components and centromeric chromatin, linker proteins such as centromere protein (CENP)-C or CENP-T in vertebrates and, additionally CENP-QOkp1-UAme1 in yeasts, also perform the function of force propagation within the kinetochore. Recent efforts have revealed an array of linker pathways strategies to effectively recruit the largely conserved outer kinetochore. In this review, we examine these linkages used to propagate force and recruit the outer kinetochore across evolution. Further, we look at their known regulatory pathways and implications on kinetochore structural diversity and plasticity.
... Results from available in vivo studies are consistent with some of the structural events described in our model. For example, the inner kinetochore proteins and CBF3 can coexist on the Cse4 nucleosome, which is coordinated by the Ctf19-Mcm21-Okp1 complex 52,53 . Also, consistent with the dissociation of Ndc10 after the formation of the centromeric nucleosome, Ndc10 is enriched at the spindle midzone in late anaphase 54 . ...
Accurate chromosome segregation relies on the specific centromeric nucleosome–kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.
... Budding yeast have 16 inner kinetochore proteins, of which only three are essential [89]. These essential proteins are CENP-Q Okp1 and CENP-U Ame1 [90], which form a dimeric subcomplex, and CENP-C Mif2 [91][92][93]. CENP-QU Okp1/Ame1 and CENP-C Mif2 bind both DNA and Mis12c MIND [94], although they cannot bind to the same Mis12c MIND simultaneously [95]. ...
The kinetochore is a complex structure whose function is absolutely essential. Unlike the centromere, the kinetochore at first appeared remarkably well conserved from yeast to humans, especially the microtubule-binding outer kinetochore. However, recent efforts towards biochemical reconstitution of diverse kinetochores challenge the notion of a similarly conserved architecture for the constitutively centromere-associated network of the inner kinetochore. This review briefly summarizes the evidence from comparative genomics for interspecific variability in inner kinetochore composition and focuses on novel biochemical evidence indicating that even homologous inner kinetochore protein complexes are put to different uses in different organisms.
... There are only two essential inner kinetochore protein complexes in budding yeast: Mif2 and OA (Okp1/Ame1) (Meeks-Wagner et al., 1986;Ortiz et al., 1999;Pot et al., 2005;Hara and Fukagawa, 2017; Table 1). Mif2, the budding yeast homolog of human CENP-C, is reported to interact with centromeric nucleosomes (Xiao et al., 2017), OA (Hornung et al., 2014), and MIND (Hornung et al., 2014;Dimitrova et al., 2016). ...
Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a ‘parts list’ of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.
... There are only two essential inner kinetochore protein complexes in budding yeast: Mif2 and OA (Okp1/Ame1) (Meeks-Wagner et al., 1986;Ortiz et al., 1999;Pot et al., 2005;Hara and Fukagawa, 2017; Table 1). Mif2, the budding yeast homolog of human CENP-C, is reported to interact with centromeric nucleosomes (Xiao et al., 2017), OA (Hornung et al., 2014), and MIND (Hornung et al., 2014;Dimitrova et al., 2016). ...
Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a ‘parts list’ of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.
... To uncover regulators of centromere function, we made use of the observation that a mutation of arginine 37 of Cse4 (cse4-R37A), which is a methylation site, causes a synthetic growth defect when combined with a mutation in the gene encoding the kinetochore component Okp1 CENP-Q [okp1-5 (42); the superscript indicates the mammalian ortholog] (6, 43) (Fig. 1A). We isolated mutations that suppressed the growth defect and identified the causative mutation by whole-genome sequencing. ...
Significance
Centromeres are the sites on the chromosome where kinetochores are assembled, a process that is required for faithful chromosome segregation. Nucleosomes in centromeric chromatin contain the histone H3 variant CENP-A. Here, we have identified the AAA ⁺ ATPase Yta7/ATAD2 as a deposition factor for CENP-A at centromeres in yeast. Our findings indicate that Yta7 acts as a hexameric AAA ⁺ ATPase that unfolds CENP-A/H4 and hands it over to Scm3/HJURP for incorporation into the centromeric nucleosome. Defects in this process result in kinetochore instability and chromosome segregation defects. The human homolog ATAD2 is frequently overexpressed in cancer cells, suggesting that it contributes to carcinogenesis by impairing chromosome segregation.
... We next examined if the N-terminus of Cse4 is required for the SDL of GAL-CSE4 in met30-6 and cdc4-1 strains. The rationale for this is based on the essential role of the N-terminus for its interactions with kinetochore proteins, Ub-mediated proteolysis and post-translational modifications (PTMs) of Cse4 [19,26,27,[54][55][56][57][58][59][60][61][62]. Furthermore, we recently showed that hir mutants, which are defective in proteolysis of overexpressed Cse4, are sensitive to GAL-CSE4 but not GAL-cse4Δ129 (Cse4 lacking the N-terminal domain) [33]. ...
Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, F-box (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of Cse4 is a major mechanism that prevents stable maintenance of Cse4 at non-centromeric regions, thus ensuring faithful chromosome segregation. In summary, we have identified essential pathways that regulate cellular levels of endogenous Cse4 and shown that proteolysis of Cse4 by SCF-Met30/Cdc4 prevents mislocalization and CIN in unperturbed cells.
... Cse4 recruitment to the centromere also depends on the CBF3 complex (Ortiz et al., 1999). Together the CBF3 complex, which is not conserved between yeast and humans, and Cse4 are required for the recruitment of all the central and outer kinetochore proteins in budding yeast. ...
The kinetochore is a large protein complex that assembles on centromeres. It enables accurate chromosome segregation by attaching chromosomes to the mitotic spindle via microtubules and by recruiting regulators that control mitosis. Many of these regulators modify kinetochore components by post-translational modifications such as phosphorylation and ubiquitination. For example, when kinetochores fail to attach to microtubules, the kinase Mps1 phosphorylates the kinetochore to activate the spindle assembly checkpoint (SAC). The abundance of chromosomal instability mutants suggests that numerous kinetochore regulators remain to be identified and the mechanisms of some known regulators are poorly characterised. The kinetochore is highly conserved from yeast to humans and many important discoveries of kinetochore function have been made using Saccharomyces cerevisiae. In order to identify kinetochore regulators in budding yeast, I have used the Synthetic Physical Interaction (SPI) technology to separately associate each member of the yeast proteome with a kinetochore protein. In addition, I have recruited several hundred proteins involved in chromosome segregation and post-translational modifications individually to kinetochore proteins representing each of the major kinetochore subcomplexes. I then associated each candidate regulator separately with most of the kinetochore proteins to spatially map their function. This method identifies candidate kinetochore regulators such as chromatin remodelling complexes, phosphatases and kinases; some of which are novel. In this thesis, I focus on three regulators; first, the Cdc14 phosphatase, second, components of the SAC and lastly the Polo-like kinase. These studies reveal kinetochore proteins sensitive to specific regulators and that their action is spatially restricted within the large kinetochore structure.
... We observed that GALCSE4 but not GALcse4∆129 results in SDL in the hir mutants. The N-terminus of Cse4 has been shown to be essential for its interactions with a subset of kinetochore proteins (ORTIZ et al. 1999;CHEN et al. 2000;MOREY et al. 2004;AU et al. 2013;HORNUNG et al. 2014). We propose that the SDL phenotype of GALCSE4 in hir mutants is partly due to titration of kinetochore proteins to ectopic sites by interactions with the N-terminal tail of mislocalized full length Cse4. ...
Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A (Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A leads to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide Synthetic Genetic Array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex (HIR1, HIR2, HIR3, HPC2) and a Cse4-specific E3 ubiquitin ligase, PSH1, showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to non-centromeric regions for genome stability. Hir2 interacts with Cse4 invivo and hir2Δ strains exhibit defects in Cse4 proteolysis, and stabilization of chromatin-bound Cse4. Mislocalization of Cse4 to non-centromeric regions with a preferential enrichment at promoter regions was observed in hir2Δ strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1 and that defects in Psh1-mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2Δ strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4 thereby promoting genome stability.
... Part of this domain is essential for centromere function, because deletion of the END region (essential N-terminal domain, amino acids 28 -60) is lethal (Chen et al. 2000). This domain recruits inner kinetochore complexes to the centromere (Ortiz et al. 1999) and possibly serves to compensate for the fact that S. cerevisiae has only one rather than multiple CENP-A/Cse4 nucleosomes at the centromere, as is the case in higher eukaryotes. ...
Centromeres are the sites of assembly of the kinetochore, which connect the chromatids to the microtubules for sister chromatid segregation during cell division. Centromeres are characterized by the presence of the histone H3 variant CENP-A (termed Cse4 in Saccharomyces cerevisiae). Here, we investigated the function of serine 33 phosphorylation of Cse4 (Cse4-S33ph) in S. cerevisiae, which lies within the essential N-terminal domain (END) of the extended Cse4 N-terminus. Significantly, we identified histone H4-K5, 8, 12R to cause a temperature-sensitive growth defect with mutations in Cse4-S33 and sensitivity to nocodazole and hydroxyurea. Furthermore, the absence of Cse4-S33ph reduced the levels of Cse4 at centromeric sequences, suggesting that Cse4 deposition is defective in the absence of S33 phosphorylation. We furthermore identified synthetic genetic interactions with histone H2A-E57A and H2A-L66A, which both cause a reduced interaction with the histone chaperone FACT and reduced H2A/H2B levels in chromatin, again supporting the notion that a combined defect of H2A/H2B and Cse4 deposition causes centromeric defects. Altogether, our data highlight the importance of correct histone deposition in building a functional centromeric nucleosome and suggests a role for Cse4-S33ph in this process.
... Cohesin is specifically enriched around the centromeres. This is due, in part, to the protein Mcm21 that facilitates SCC around the centromere in a manner yet to be determined (Ortiz et al. 1999;Poddar et al. 1999;Ng et al. 2009). The centromere enrichment of cohesin facilitates sister chromatid biorientation before mitosis (Ng et al. 2009;Stephens et al. 2011Stephens et al. , 2013, assuring proper chromatid segregation, and the prevention of aneuploidy. ...
Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.
... GIS ≤ -25) and five out of the six strongest enhancers were members of the Ctf19 complex (Table S5). Only MCM21 and CTF19, encoding the two non-essential members of the COMA sub-complex (Ame1 and Okp1 are both essential: De Wulf et al. 2003;Ortiz et al. 1999),! were amongst the strongest enhancer of bir1-17 at all three screening temperatures. ...
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay (NMD) pathway caused strong phenotypic suppression. Thus cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome bi-orientation. Loss of some Ctf19 components such as Iml3 or Chl4 impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3 Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.
... The first centromere-specific kinetochore proteins were discovered in clinical studies performed from patients with progressive systemic sclerosis (CREST syndrome) [32]. Advances in proteo mics have enabled the identification of a large number of kinetochore components [98][99][100][101][102]. However, functions of the many kinetochore components have been well worked out, little is known about how they are recruited to the centromere or how they assemble to form the complex kinetochore structure. ...
In most metazoans, multiple microtubules bind to each kinetochore, with an exception of certain budding yeasts where only a single microtubule appears to be associated with each kinetochore. Apart from these general features of mitosis, organism-specific variations also exist. During closed mitosis, the nuclear envelope (NE) remains intact throughout the cell cycle, the spindle forms within the nucleus followed by chromosomal segregation and subsequent nuclear fission. This chapter discusses players and the process of chromosome segregation via mitotic cell cycle in fungal, animal, and plant kingdoms. It also discusses the growing knowledge of the same in protozoa as well. The fungal kinetochores are well studied and formed by more than 80 known proteins assembled on the centromere DNA. Molecular details of the proteins and their organization in a plant kinetochore are less explored compared to other eukaryotes.
... Most of the CCAN subunits have orthologs in S. cerevisiae [107,114,115,[117][118][119], which are collectively identified as the Ctf19 complex ( Figure 2A). A notable exception is the 4-subunit CBF3 complex ( Figure 2B,C), a cognate binding partner of the CEN DNA of S. cerevisiae [34]. ...
Kinetochores are large protein assemblies that connect chromosomes to microtubules of the mitotic and meiotic spindles in order to distribute the replicated genome from a mother cell to its daughters. Kinetochores also control feedback mechanisms responsible for the correction of incorrect microtubule attachments, and for the coordination of chromosome attachment with cell cycle progression. Finally, kinetochores contribute to their own preservation, across generations, at the specific chromosomal loci devoted to host them, the centromeres. They achieve this in most species by exploiting an epigenetic, DNA-sequence-independent mechanism; notable exceptions are budding yeasts where a specific sequence is associated with centromere function. In the last 15 years, extensive progress in the elucidation of the composition of the kinetochore and the identification of various physical and functional modules within its substructure has led to a much deeper molecular understanding of kinetochore organization and the origins of its functional output. Here, we provide a broad summary of this progress, focusing primarily on kinetochores of humans and budding yeast, while highlighting work from other models, and present important unresolved questions for future studies.
... mutants that have increased rates of chromosome loss [32,33], one-hybrid [34] and two-hybrid screens [35] all led to the identification of many kinetochore proteins. More recently, affinity purification and mass spectrometry analysis accelerated the identification [36,37]. ...
The kinetochore is the macromolecular protein complex that drives chromosome segregation in eukaryotes. Its most fundamental function is to connect centromeric DNA to dynamic spindle microtubules. Studies in popular model eukaryotes have shown that centromere protein (CENP)-A is critical for DNA-binding, whereas the Ndc80 complex is essential for microtubule-binding. Given their conservation in diverse eukaryotes, it was widely believed that all eukaryotes would utilize these components to make up a core of the kinetochore. However, a recent study identified an unconventional type of kinetochore in evolutionarily distant kinetoplastid species, showing that chromosome segregation can be achieved using a distinct set of proteins. Here, I review the discovery of the two kinetochore systems and discuss how their studies contribute to a better understanding of the eukaryotic chromosome segregation machinery.
... proteins that localize to the centromere throughout the cell cycle . Ortiz et al., 1999). In mammals, CENP-U is essential for early mouse development (Kagawa et al., 2014), but eliminating CENP-U and CENP-Q results in relatively mild phenotypes in tissue culture cells Kagawa et al., 2014). ...
Each time a cell divides, the genome must be segregated equally between the two new daughter cells. To accomplish this, a specific region of each chromosome, termed the centromere, recruits the macromolecular kinetochore structure to mediate attachments to spindle microtubules. In vertebrates, each chromosome must establish a single site of microtubule attachment. The failure to maintain this site or the generation of multiple distinct microtubule attachment sites on a single chromosome can have profoundly deleterious effects on cell and organismal viability. My graduate work has used cell biological analyses in tissue culture cells and biochemical reconstitutions to define the molecular mechanisms by which human cells maintain one and only one site of microtubule attachment on each chromosome. First, I defined the regulatory paradigms that ensure the faithful propagation of the centromere. I identified Polo-like kinase 1 as a key player in controlling the deposition of the epigenetic mark that specifies the centromere, the CENP-A nucleosome. I defined the molecular basis for this control, as well as an additional level of control downstream of the cyclin-dependent kinases. By identifying and dissecting the molecular features of this two-step regulatory paradigm, I developed a strategy to bypass the control of CENP-A deposition, which resulted in severe mitotic defects. In my second project, I defined the architecture and properties of the sixteen-protein assembly that connects CENP-A to the other proteins of the kinetochore. I analyzed the genetic relationships between these proteins in human cells through a combination of inducible knockouts and inducible protein degradation. I then reconstituted the sixteen proteins in vitro as five sub-complexes and defined their interactions biochemically. These analyses revealed an intricate meshwork of direct interactions between the proteins at the centromere-kinetochore interface, which is critical for ensuring assembly of the kinetochore at the correct site on the chromosome. Together, these findings provide new insights into the molecular mechanisms of centromere propagation and kinetochore assembly.
... Constructs of PGAL-CEN3-tetOs (Tanaka et al., 2005), TetR-3CFP (Bressan et al., 2004), PMET3-CDC20 (Uhlmann et al., 2000), TetR-GFP (Michaelis et al., 1997) and REC102-lacOs (Straight et al., 1996;Sullivan et al., 2004) were as described previously. Temperature-sensitive mutants ndc10-1 (Goh and Kilmartin, 1993]), okp1-5 (Ortiz et al., 1999), dsn1-7 (Nekrasov et al., 2003), spc24-1 (Wigge and Kilmartin, 2001), dam1-1 (Cheeseman et al., 2001]), ipl1-321 (Biggins et al., 1999), tub4-1 , spc98-1 , stu2-10 (Severin et al., 2001) were as described previously. To make bim1, bik1 and kar3, whole open reading frames of the relevant genes were replaced with antibiotic-resistance genes, using a one-step PCR method (Amberg et al., 2005). ...
... The linker layer serves as a bridge between the centromere-binding proteins of the inner KT and the MT-binding proteins of the outer KT (Westermann, S. et al., 2007). A protein complex that is also counted to the CCAN, but is also part of the linker layer is the COMA complex containing of the proteins Ctf19, Okp1, Mcm21 and Ame1 (Ortiz, J. et al., 1999;De Wulf, P. et al., 2003). Various additional proteins that bind to the CO-MA complex are taken together as the Ctf19 complex in budding yeast and are also counted among the CCAN. ...
The kinetochore (KT) is a complex structure that enables attachment of chromosomes to spindle microtubules (MTs). Several MT associated proteins (MAPs) contribute to the KT-MT interface and regulate the dynamics of kinetochore microtubules (kMTs). In addition, these MAPs localize to interpolar MTs and regulate spindle stability. One of these proteins is the S. cerevisiae CLASP (cytoplasmic linker associated protein) Stu1, an essential protein that has several functions during mitosis and therefore localizes differently in the course of each cell division. The aim of this work was to investigate which domains of Stu1 are important for its cell cycle specific localization, how this contributes to a coordinated action of Stu1 and how localization and function of Stu1 are regulated. Structural predictions of Stu1 suggest the organization in six domains. The following observations were made with the focus on three of them: the TOGL2 domain, the minimal MT-binding loop (ML) and the C-terminal loop (CL).
The TOGL2 domain solely achieves binding of αβ-tubulin and drives spindle formation
Co-immunoprecipitations identified the TOGL2 domain to be sufficient to bind free αβ-tubulin. This feature of the TOGL2 domain is essential and solely responsible for the important role of Stu1 in driving spindle formation. Thus, the TOGL2 domain ensures the function of Stu1 as a MT polymerase or rescue factor. Domain swapping experiments demonstrated that the function of the TOGL2 domain of Stu1 is very specific and cannot be easily taken over by another TOG domain.
MT binding via the ML domain is required for efficient metaphase spindle formation, but is dispensable for midzone localization
Besides the TOGL2 domain, efficient spindle formation in metaphase additionally depends on the binding of Stu1 to the MT lattice via the ML domain. Thereby, the CL domain specifies Stu1 localization to the region of the MT overlap. Midzone localization of Stu1 in anaphase, however, is independent of the ML domain and therefore must be ensured in a manner that is not based on MT binding.
An interplay of the CL domain with the ML domain specifies Stu1’s sequestration at unattached KTs
The CL domain was found to specify Stu1 for the sequestration at unattached KTs, most likely by inhibiting the MT binding affinity of the ML domain. Thus, the CL domain indirectly prevents spindle formation in the presence of unattached KTs.
Efficient KT capture relies on unperturbed MT dynamics ensured by the Stu1 TOGL2 activity
Capturing experiments of the CL deletion mutant revealed that Stu1 localization to unattached KTs is not a prerequisite for efficient capturing. However, the ML domain and especially the TOGL2 activity are mandatory in this respect. The contribution of Stu1 to unperturbed MT dynamics seems to be more important for the capturing pro-cess than KT localization. This may involve the polymerization of capturing kMTs but also, as analyzed, the temporal regulation of KT-generated MTs.
The CL domain makes kMT length dependent on the tension on the KT-MT inter-face
The localization of Stu1 to attached KTs is a prerequisite for the polymerization of kMTs. In this respect, the CL domain was found to inhibit Stu1’s ability to stabilize kMTs and to make the kMT length dependent on tension on the KT-MT interface.
The CL domain prevents precocious spindle formation to ensure biorientation
The data showed that the CL domain facilitates bipolar attachment by ensuring unperturbed dynamics of interpolar MTs. Therefore, the CL domain seems to fine-tune the MT polymerizing activity of Stu1 by regulating the MT affinity of the ML domain.
Stu1 phosphorylation within the CL domain contributes to Stu1 regulation
Finally, this work revealed that phosphorylation of Stu1 contributes to the regulation of Stu1. SILAC analyses identified 15 phosphorylation sites that mainly reside within the ML and the CL domain of Stu1 and are putative target sites of various serine/ threonine kinases like Cdk1, polo-like kinase, Ipl1 and Mps1. Furthermore, these analyses demonstrated that Stu1 gets phosphorylated and dephosphorylated throughout the cell cycle suggesting a regulatory role for kinases. Consistent with that, in vitro kinase as-says identified Stu1 N- and C-terminus as targets of Ipl1 and Mps1 kinases. Analyses of phosphomutants eventually suggested that phosphorylation of the CL domain con-tributes to the regulatory impact of the CL domain on the MT affinity of the ML domain.
Taken together, the present study supports the theory that Stu1, similar to other CLASP proteins acts as a local modulator for MT dynamics and stability. While the TOGL2 domain accomplishes the essential function of tubulin incorporation in MT plus-ends, the other domains are required to regulate the localization of Stu1 and (probably therefore) control the MT polymerizing activity.
Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP‐A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP‐A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1‐Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.
Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for β-1,6- and β-1,3 glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/transglycosidase required for β-1,6 glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a CCAN protein. Similarly, the absence of Fks1, a β-1,3 glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular β-1,6 and β -1,3 glucan, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.
Kinetochores are large protein complexes built on centromeric chromatin that mediate chromosome segregation. The inner kinetochore, or constitutive centromere-associated network (CCAN), assembles onto centromeres defined by centromere protein A (CENP-A) nucleosomes (CENP-ANuc), and acts as a platform for the regulated assembly of the microtubule-binding outer kinetochore. Recent cryo-EM work revealed structural conservation of CCAN, from the repeating human regional centromeres to the point centromere of budding yeast. Centromere recognition is determined mainly through engagement of duplex DNA proximal to the CENP-A nucleosome by a DNA-binding CENP-LN channel located at the core of CCAN. Additional DNA interactions formed by other CCAN modules create an enclosed DNA-binding chamber. This configuration explains how kinetochores maintain their tight grip on centromeric DNA to withstand the forces of chromosome segregation. Defining the higher-order architecture of complete kinetochore assemblies with implications for understanding the 3D organisation of regional centromeres and mechanisms of kinetochore dynamics, including how kinetochores sense and respond to tension, are important future directions.
Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.
Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.
Kinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affects accurate chromosome segregation in a Sli15 wild-type background. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere inner kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating accurate chromosome segregation.
During a single human lifetime, nearly one quintillion chromosomes separate from their sisters and transit to their destinations in daughter cells. Unlike DNA replication, chromosome segregation has no template, and, unlike transcription, errors frequently lead to a total loss of cell viability. Rapid progress in recent years has shown how kinetochores enable faithful execution of this process by connecting chromosomal DNA to microtubules. These findings have transformed our idea of kinetochores from cytological features to immense molecular machines and now allow molecular interpretation of many long-appreciated kinetochore functions. In this review we trace kinetochore protein connectivity from chromosomal DNA to microtubules, relating new findings to important points of regulation and function.
A number of paths have led to the present list of centromere proteins, which is essentially complete for constitutive structural proteins, but still may be only partial if we consider the many other proteins that briefly visit the centromere and kinetochore to fine-tune the chromatin and adjust other functions. Elegant genetics led to the description of the budding yeast point centromere in 1980. In the same year was published the serendipitous discovery of antibodies that stained centromeres of human mitotic chromosomes in antisera from CREST patients. Painstaking biochemical analyses led to the identification of the human centromere antigens several years later, with the first yeast proteins being described 6 years after that. Since those early days, the discovery and cloning of centromere and kinetochore proteins has largely been driven by improvements in technology. These began with expression cloning methods, which allowed antibodies to lead to cDNA clones. Next, functional screens for kinetochore proteins were made possible by the isolation of yeast centromeric DNAs. Ultimately, the completion of genome sequences for humans and model organisms permitted the coupling of biochemical fractionation with protein identification by mass spectrometry. Subsequent improvements in mass spectrometry have led to the current state where virtually all structural components of the kinetochore are known and where a high-resolution map of the entire structure will likely emerge within the next several years.
SUMO E3 ligases are known to have a major role in preventing gross chromosomal rearrangements (GCRs); however, relatively little is known about the role of SUMO isopeptidases in genome maintenance and their role in controlling intracellular sumoylation homeostasis. Here we show the SUMO isopeptidase Ulp2 in Saccharomyces cerevisiae does not prevent the accumulation of GCRs and interestingly, its loss causes subunit-specific changes of sumoylated MCM helicase in addition to drastic accumulation of sumoylated nucleolar RENT and inner kinetochore complexes. In contrast, loss of Ulp1 or its mis-localization from the nuclear periphery causes substantial accumulations of GCRs and elevated sumoylation of most proteins except for Ulp2 targets. Interestingly, the E3 ligase Mms21, which has a major role in genome maintenance, preferentially controls the sumoylation of Mcm3 during DNA replication. These findings reveal distinct roles for Ulp1 and Ulp2 in controlling homeostasis of intracellular sumoylation and show that sumoylation of MCM is controlled in a subunit-specific and cell cycle dependent manner.
The somatic division, called mitosis, is characterized by equal distribution of parental genome into two daughter cells. Mitosis involves a dramatic reorganization of both nucleus and cytoplasm driven by protein kinase cascades including master controller Cdkl-cyclin B. Mitosis is an ancient eukaryotic event, and some divergence emerged during evolution. Many single cell eukaryotes, including yeast and slime molds, undergo a closed mitosis, in which mitotic spindle formation and chromosome segregation occur within an intact nuclear envelope. However, higher eukaryotes such as animal and plant cells use open mitosis, in which nuclear envelope disassembles before the chromosomes segregate. This review primarily focuses on mitotic chromosome segregation in animal cells and refers to other organisms when regulation is mechanistically conserved. For convenience of discussion, mitotic chromosome dynamics are subdivided into six phases: prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis.
The centromere is the region of the chromosome that directs its segregation in mitosis and meiosis. Although the functional importance of the centromere has been appreciated for more than 130 years, elucidating the molecular features and properties that enable centromeres to orchestrate chromosome segregation is an ongoing challenge. Most eukaryotic centromeres are defined epigenetically and require the presence of nucleosomes containing the histone H3 variant centromere protein A (CENP-A; also known as CENH3). Ongoing work is providing important molecular insights into the central requirements for centromere identity and propagation, and the mechanisms by which centromeres recruit kinetochores to connect to spindle microtubules.
Centromeres are the differentiated chro-mosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of eentromeric chromatin, we have cloned a human eDNA that encodes the 17-kD historic-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of cen-tromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.
We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).
Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200
base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core
and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the
sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit
altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional
centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion
by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted
in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic
attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding
CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes
may be the central core of the centromere itself.
Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.
We have designed a screen to identify mutants specifically affecting kinetochore function in the yeast Saccharomyces cerevisiae. The selection procedure was based on the generation of "synthetic acentric" minichromosomes. "Synthetic acentric" minichromosomes contain a centromere locus, but lack centromere activity due to combination of mutations in centromere DNA and in a chromosomal gene (CEP) encoding a putative centromere protein. Ten conditional lethal cep mutants were isolated, seven were found to be alleles of NDC10 (CEP2) encoding the 110-kD protein of yeast kinetochore. Three mutants defined a novel essential gene CEP3. The CEP3 product (Cep3p) is a 71-kD protein with a potential DNA-binding domain (binuclear Zn-cluster). At nonpermissive temperature the cep3 cells arrest with an undivided nucleus and a short mitotic spindle. At permissive temperature the cep3 cells are unable to support segregation of minichromosomes with mutations in the central part of element III of yeast centromere DNA. These minichromosomes, when isolated from cep3 cultures, fail to bind bovine microtubules in vitro. The sum of genetic, cytological and biochemical data lead us to suggest that the Cep3 protein is a DNA-binding component of yeast centromere. Molecular mass and sequence comparison confirm that Cep3p is the p64 component of centromere DNA binding complex Cbf3 (Lechner, 1994).
Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.
Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.
The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.
We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.
A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast. ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle. This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD. NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J. Lechnermn and J. Carbon. 1993. J. Cell Biol. 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J. Carbon. 1991. Cell. 61:717-725). NDC10 is an essential gene. Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells. In some cells with short spindles which may be in metaphase, staining was also observed along the spindle. The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.
A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants
have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments
allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation.
Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex.
Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally
impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control
assay has allowed assembly steps preceding chromosome segregation to be defined.
We have designed and utilized two in vivo assays of kinetochore integrity in S. cerevisiae. One assay detects relaxation of a transcription block formed at centromeres; the other detects an increase in the mitotic stability of a dicentric test chromosome. ctf13-30 and ctf14-42 were identified as putative kinetochore mutants by both assays. CTF14 is identical to NDC10/CBF2, a recently identified essential gene that encodes a 110 kd kinetochore component. CTF13 is an essential gene that encodes a predicted 478 amino acid protein with no homology to known proteins. ctf13 mutants missegregate chromosomes at permissive temperature and transiently arrest at nonpermissive temperature as large-budded cells with a G2 DNA content and a short spindle. Antibodies recognizing epitope-tagged CTF13 protein decrease the electrophoretic mobility of a CEN DNA-protein complex formed in vitro. Together, the genetic and biochemical data indicate that CTF13 is an essential kinetochore protein.
Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.
Kinetochores are DNA-protein structures that attach and move chromosomes along the microtubules of the mitotic spindle. This review focuses on centromeres and kinetochores from the budding yeast Saccharomyces cerevisiae. Because of their relative simplicity, budding yeast centromeres are particularly well suited to detailed molecular analysis. The review begins with a description of chromosome movement and microtubule attachment in living yeast cells. We then describe how centromeric DNA was cloned and analyzed and how this analysis has led to the isolation of centromere-binding proteins by both biochemical and genetic means. Modes of microtubule attachment in vitro are discussed with an attempt to relate in vitro studies to the events that occur in vivo. Finally, we conclude with a speculative model for the organization of the budding yeast centromere a consideration of possible future developments.
The budding yeast SKP1 gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro. Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes. skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function. skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase. Identification of Skp1p homologs from C. elegans, A. thaliana, and H. sapiens indicates that SKP1 is evolutionarily highly conserved. Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery.
We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations. To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26. ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1. These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants. In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.
The centromere is an essential structure on all eukaryotic chromosomes that allows the equipartition of chromosomes during mitotic and meiotic cell divisions. Since its cytogenetic recognition as a constructed part of a chromosome many decades ago, great advance has been made on our understanding of this intriguing structure, especially at the molecular level. This book brings together all available information on the centromere. It covers in details the DNA and protein components of this structure, and their individual functions, in species as diverse as budding and fission yeasts, nematodes, Drosophila, mice, and humans; newly discovered roles of the centromere in marshalling "passenger" proteins; important emerging concepts such as latest centromeres and epigenetic factors; cytogenetic problems associated with centromere abnormalities; and practical application of centromere studies, such as in the construction of human artificial chromosomes for gene therapy. Supported by ample illustration, the book is written with sufficient simplicity and detail to suit both specialist and non-specialist scholars. It is the first book on the subject
Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.
The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.
Kinetochores are structures that assemble on centromeric DNA and mediate the attachment of chromosomes to the microtubules of the mitotic spindle. The protein components of kinetochores are poorly understood, but the simplicity of the S. cerevisiae kinetochore makes it an attractive candidate for molecular dissection. Mutations in genes encoding CBF1 and CBF3, proteins that bind to yeast centromeres, interfere with chromosome segregation in vivo. To determine the roles played by these factors and by various regions of centromeric DNA in kinetochore function, we have developed a method to partially reassemble kinetochores on exogenous centromeric templates in vitro and to visualize the attachment of these reassembled kinetochore complexes to microtubules. In this assay, single reassembled complexes appear to mediate microtubule binding. We find that CBF3 is absolutely essential for this attachment but, contrary to previous reports (Hyman, A. A., K. Middleton, M. Centola, T.J. Mitchison, and J. Carbon. 1992. Microtubule-motor activity of a yeast centromere-binding protein complex. Nature (Lond.). 359:533-536) is not sufficient. Additional cellular factors interact with CBF3 to form active microtubule-binding complexes. This is mediated primarily by the CDEIII region of centromeric DNA but CDEII plays an essential modulatory role. Thus, the attachment of kinetochores to microtubules appears to involve a hierarchy of interactions by factors that assemble on a core complex consisting of DNA-bound CBF3.
We have cloned and determined the nucleotide sequence of the gene (CBF2) specifying the large (110 kD) subunit of the 240-kD multisubunit yeast centromere binding factor CBF3, which binds selectively in vitro to yeast centromere DNA and contains a minus end-directed microtubule motor activity. The deduced amino acid sequence of CBF2p shows no sequence homologies with known molecular motors, although a consensus nucleotide binding site is present. The CBF2 gene is essential for viability of yeast and is identical to NDC10, in which a conditional mutation leads to a defect in chromosome segregation (Goh, P.-Y., and J. V. Kilmartin, in this issue of The Journal of Cell Biology). The combined in vitro and in vivo evidence indicate that CBF2p is a key component of the budding yeast kinetochore.
The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.
Temporal control of ubiquitin-proteasome mediated protein degradation is critical for normal G1 and S phase progression. Recent work has shown that central to the temporal control mechanism is a relationship between newly identified E3 ubiquitin protein ligases, designated SCFs (Skp1-cullin-F-box protein ligase complexes), which confer substrate specificity on ubiquitination reactions and the activities of protein kinases that phosphorylate substrates destined for destruction at specific sites, thereby converting them into preferred targets for ubiquitin modification catalyzed by SCFs. The constituents of SCFs are members of evolutionary conserved protein families. SCF-based ubiquitination pathways may play a key role in diverse biological processes, such as cell proliferation, differentiation and development.
We have developed a simple procedure for the localized mutagenesis of yeast genes. In this technique the region of interest is first amplified under mutagenic polymerase chain reaction (PCR) conditions. Cotransformation of the PCR product with a gapped plasmid containing homology to both ends of the PCR product allows in vivo recombination to repair the gap with the mutagenized DNA. This procedure is efficient, allows targeting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.
A key protein component (CBF3) of the budding yeast (S. cerevisiae) centromere/kinetochore has been purified and characterized. CBF3 is a 240 kd multisubunit protein complex that binds specifically to the yeast wild-type centromere DNA (CEN), but not to nonfunctional CEN DNA containing a single base substitution in the critical CDEIII consensus sequence. When purified by affinity chromatography, CBF3 contains three protein components: CBF3A (110 kd), CBF3B (64 kd), and CBF3C (58 kd). Highly purified CBF3 requires the presence of a separate assembly factor or chaperone activity to bind to CEN DNA. Treatment with phosphatase inactivates CBF3, indicating that at least one of the CBF3 subunits must be phosphorylated for DNA binding to occur. A 56 bp region including the 26 bp CDEIII consensus is protected from DNAase I cleavage in the CBF3-CEN DNA complex.
We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis. Eighty-five percent of the mutations are recessive and 15% are partially dominant. Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1. For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type. The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations. Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees. In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype. Five additional complementation groups (ctf12 through ctf16) have been defined by complementation analysis of the mutations causing inviability at 37 degrees. Twenty-three of the 136 isolates exhibited growth defects at concentrations of benomyl permissive for the parent strain, and nine appeared to be tolerant of inhibitory levels of benomyl. All of the mutant strains showed normal sensitivity to ultraviolet and gamma-irradiation. Further characterization of these mutant strains will describe the functions of gene products crucial to the successful execution of processes required for aspects of the chromosome cycle that are important for chromosome transmission fidelity in mitosis.
Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional
centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III,
a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region
have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been
confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally
altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element
III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters.
Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays.
The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in
the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.
In Saccharomyces cerevisiae, 3-amino-1,2,4-triazole (aminotriazole) competitively inhibits the activity of imidazoleglycerolphosphate dehydratase, the product of the HIS3 gene. Wild-type strains are able to grow in the presence of 10 mM aminotriazole because they induce the level of imidazoleglycerolphosphate dehydratase. However, strains containing gcn4 mutations are unable to grow in medium containing aminotriazole because they lack the GCN4 transcriptional activator protein necessary for the coordinate induction of HIS3 and other amino acid biosynthetic genes. Here, we isolated a new gene, designated ATR1, which when present in multiple copies per cell allowed gcn4 mutant strains to grow in the presence of aminotriazole. In wild-type strains, multiple copies of ATR1 permitted growth at extremely high concentrations of aminotriazole (80 mM), whereas a chromosomal deletion of ATR1 caused growth inhibition at very low concentrations (5 mM). When radioactive aminotriazole was added exogenously, cells with multiple copies of ATR1 accumulated less aminotriazole than wild-type cells, whereas cells with the atr1 deletion mutation retained more aminotriazole. Unlike the mammalian mdr or yeast PDR genes that confer resistance to many drugs, ATR1 appears to confer resistance only to aminotriazole. Genetic analysis, mRNA mapping, and DNA sequencing revealed that (i) the primary translation product of ATR1 contains 547 amino acids, (ii) ATR1 transcription is induced by aminotriazole, and (iii) the ATR1 promoter region contains a binding site for the GCN4 activator protein. The deduced amino acid sequence suggests that ATR1 protein is very hydrophobic with many membrane-spanning regions, has several potential glycosylation sites, and may contain an ATP-binding site. We suggest that ATR1 encodes a membrane-associated component of the machinery responsible for pumping aminotriazole (and possibly other toxic compounds) out of the cell.
A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.
This chapter discusses the visual assay for chromosome ploidy. The chapter describes the ade3-2p and SUPII visual assays for following changes in chromosome ploidy during mitotic divisions of S.cerevisiae. The chapter presents three experimental applications of the assays: the sectoring assay, a qualitative assay for identifying cis and trans mutations that show an altered fidelity of chromosome transmission; fluctuation analysis, a quantitative method for determining changes in the rate of chromosome loss or gain; and half-sectoring colony analysis, an assay for determining the types of aberrant transmissions that are responsible for producing cells with altered chromosome ploidy. The chapter describes the general principles and the materials and reagents necessary to perform each application and the sample experiments, which demonstrate their use and limitations in the isolation and characterization of mutants affecting chromosome transmission. The sample experiments use the ade3-2p system.
Two DNA sequences that reduce mitotic fidelity of chromosome transmission have been identified: MIF1 and MIF2. MIF1 is a unique sequence located on the right arm of chromosome XII that stimulates loss and recombination for both chromosomes V and VII when present in a high copy number plasmid. MIF1 is not essential for cell division but is necessary for the normal fidelity of chromosome transmission. MIF2 is a unique sequence located 15 cM distal to HIS6 on chromosome IX that induces a high frequency of chromosome VII loss and a lower frequency of chromosome V loss when present in high copy number; it has no effect on mitotic recombination. Disruption of the genomic MIF2 locus was lethal and cells lacking this function arrested division with a terminal phenotype characteristic of a block in DNA replication or nuclear division.
We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.
The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC10/CBF2, and CEP3/CBF3B. In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEP1. Synthetic lethal interactions between a cep1 null mutation and mutations in either NDC10 or CEP3 were also detected. Taken together, these data suggest that the Mif2 protein interacts with Cep1p at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex. The Mif2 and Cep1 proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication. We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions. These results provide evidence for structural conservation between yeast and mammalian centromeres.
A short stretch of strong homology between the Saccharomyces cerevisiae chromosome segregation protein Mif2 and the DNA-binding motifs of the Drosophila D1 and mammalian HMGI(Y) chromosomal proteins suggested that Mif2 may act directly on chromosomes. Because this conserved motif is involved in binding A.T DNA, it was proposed that Mif2 may interact with chromosomes at the highly A + T-rich DNA element found in yeast centromeres. Comparison of the Mif2 amino-acid sequence with sequence databases showed that Mif2 shares at least two regions of similarity with the mammalian centromere protein CENP-C, suggesting an evolutionary conservation of centromere protein function from yeast to mammals. The order, spacing and location of these regions are also similar in the two proteins. Sequence analysis of several conditional lethal alleles of MIF2 generated by random mutagenesis revealed mutations in regions homologous to CENP-C, as well as in the highly conserved A.T DNA-binding motif. A potential phosphorylation site for p34cdc2 kinase located adjacent to the A.T DNA-binding motif was also found to be mutated in one of the mutants, suggesting that phosphorylation at this site may be important for Mif2 function and possibly for DNA binding.
Regulation of transcription involves the assembly of multiprotein complexes at enhancers and promoters. Interactions between adjacent and non-adjacent DNA-binding proteins can augment the specificity and stability of multi-component nucleoprotein complexes. Recently, several proteins have been identified that can function as 'architectural' elements in the assembly of higher-order nucleoprotein structures reminiscent of those involved in site-specific recombination in prokaryotes.
The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.
An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The KM-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The VMAX but not the KM-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS-polyacrylamide gels.
A multisubunit protein complex, Cbf3, is a component of the Saccharomyces cerevisiae kinetochore. Cbf3 was recently shown to be essential for chromosome segregation in vivo and for movement of centromere DNA (CEN) along microtubules in vitro. Cbf3 contains three proteins, Cbf3a, Cbf3b and Cbf3c. Here the characterization of Cbf3b is described. Cbf3b contains an N-terminal Zn2Cys6 type zinc finger domain, a C-terminal acidic domain and a putative coiled coil dimerization domain. Cbf3b is essential for growth. Mutations within the zinc finger domain result in cells that exhibit a G2-M cell cycle delay and increased chromosome loss in each mitotic cell division. Therefore, Cbf3b has an essential function in chromosome segregation and the zinc finger domain executes part of this function presumably by providing the specific interaction between Cbf3 and CEN. Finally, data are provided to show that Cbf3c is encoded by CTF13, a gene that had been cloned recently by complementing a temperature sensitive mutant that exhibits chromosome loss as a result of a defective centromere.
Processing of DNA damage by the nucleotide-excision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specifically interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that mediate this interaction. These results suggest that XPA may play a role in the localization or loading of an incision complex, composed of ERCC1 and possibly other repair factors, onto a damaged site.
The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.
Here a method is described to identify genes encoding proteins that recognize a specific DNA sequence. A bank of random protein
segments tagged with a transcriptional activation domain is screened for proteins that can activate a reporter gene containing
the sequence in its promoter. This strategy was used to identify an essential protein that interacts in vivo with the yeast
origin of DNA replication. Matches between its predicted amino acid sequence and peptide sequence obtained from the 50-kilodalton
subunit of the yeast origin recognition complex (ORC) established that the gene isolated here, ORC6, encodes this subunit.
These observations provide evidence that ORC recognizes yeast replication origins in vivo.
Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in the cell cycle.
We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.
Accurate chromosome segregation is dependent on a specialized chromosomal structure, the kinetochore/centromere. The only essential constituent of the S. cerevisiae kinetochore established today is CBF3, a multisubunit complex that binds to S. cerevisiae centromere DNA. Therefore CBF3 and its four components, Cbf3a, Cbf3b, Cbf3c and Cbf3d, will form the centerpiece of this review. In addition, we will describe proteins that are putatively involved in kinetochore function specifically in the context with CBF3 interaction. Furthermore, we discuss the role of the S. cerevisiae kinetochores in a putative cell cycle checkpoint control and in microtubule attachment.
We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.