Molecular structure of human geminin
ABSTRACT The origin licensing repressor geminin is a unique bifunctional protein providing a molecular link between cellular proliferation, differentiation and genomic stability. Here we report the first molecular structure of human geminin, determined by EM and image processing at a resolution of 17.5 A. The geminin molecule is a tetramer formed by two dimers with monomers interacting via coiled-coil domains. The unusual structural organization of geminin provides molecular insight into its bifunctional nature.
Full-textDOI: · Available from: Andrei L Okorokov, Apr 24, 2014
- SourceAvailable from: onlinelibrary.wiley.com
[Show abstract] [Hide abstract]
- "The decreased ability of mutant geminin to induce foci formation and to tether Cdt1 molecules is consistent with the decreased strength of IOC predicted by the gradual-type inhibition of origin licensing. Geminin has been reported to form different types of oligomers including dimers (Benjamin et al. 2004), dimer–dimers (Thepaut et al. 2004) and tetramers (Okorokov et al. 2004). Geminin also forms different stoichiometric complexes with Cdt1 (Lee et al. 2004; Lutzmann et al. 2006; De Marco et al. 2009). "
ABSTRACT: In metazoans, geminin functions as a molecular switch for preventing re-replication of chromosomal DNA. Geminin binds to and inhibits Cdt1, which is required for replication origin licensing, but little is known about the mechanisms underlying geminin's all-or-none action in licensing inhibition. Using Xenopus egg extract, we found that the all-or-none activity correlated with the formation of Cdt1 foci on chromatin, suggesting that multiple Cdt1-geminin complexes on origins cooperatively inhibit licensing. Based on experimental identification of licensing intermediates targeted by geminin and Cdt1, we developed a mathematical model of the licensing process. The model involves positive feedback owing to the cooperative action of geminin at neighboring origins and accurately accounts for the licensing activity mediated by geminin and Cdt1 in the extracts. The model also predicts that such cooperativity leads to clustering of licensing-inhibited origins, an idea that is supported by the experimentally measured distribution of inter-origin distances. We propose that geminin inhibits licensing through an inter-origin interaction, ensuring strict and coordinated control of multiple replication origins on chromosomes.Genes to Cells 04/2011; 16(4):380-96. DOI:10.1111/j.1365-2443.2011.01501.x · 2.86 Impact Factor
[Show abstract] [Hide abstract]
- "A collection of 5909 images from individual polymerase complexes was extracted, a fraction of which were obtained after tilting the specimen holder to help increase the angular coverage in the different views of the protein (see a gallery of single images in Figure 3B). Processing of EM images derived from small asymmetric proteins still represents an important challenge (23) but advances in the methodology has allowed some proteins in the 100-kDa range to be solved at medium resolution with the help of staining agents (38,39). We have found that, because of the absence of symmetry and the small size of the molecules, the initial reference for refinement can introduce bias during the reconstruction procedure and lead towards a local minimum. "
ABSTRACT: The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.Nucleic Acids Research 02/2007; 35(11):3774-83. DOI:10.1093/nar/gkm336 · 9.11 Impact Factor
[Show abstract] [Hide abstract]
- "The central portion of geminin contains five heptad amino acid repeats that are predicted to form a coiled-coil domain, a structure commonly associated with protein dimerization (Benjamin et al . 2004; Okorokov et al . 2004). The geminin dimer binds to Cdt1 with high affinity in the nucleus and prevents it from contributing to pre-RCs during S and G 2 phases (Wohlschlegel et al . 2000; Tada et al . 2001). Furthermore, access of the MCM complex to Cdt1 is restricted by the COOH-terminal portion of the coiled-coil domain of geminin (Lee et al . 2004). These "
ABSTRACT: Replication of DNA is strictly controlled to ensure that it occurs only once per cell cycle. Geminin has been thought to serve as a central mediator of this licensing mechanism by binding to and antagonizing the function of Cdt1 and thereby preventing re-replication during S and G2 phases. We have now generated mice deficient in geminin to elucidate the physiologic role of this protein during development. Lack of geminin was shown to result in preimplantation mortality. A delay in the development of homozygous mutant embryos was first apparent at the transition from the four- to eight-cell stages, concomitant with the disappearance of maternal geminin protein, and development was arrested at the eight-cell stage. The mutant embryos manifest morphological abnormalities such as dispersed blastomeres with nuclei that are irregular both in size and shape as well as impaired cell-cell adhesion. DNA replication occurs but mitosis was not detected in the mutant embryos. The abnormal blastomeres contain damaged DNA and undergo apoptosis, likely as a consequence of the deregulation of DNA replication. Our results suggest that geminin is essential for cooperative progression of the cell cycle through S phase to M phase during the preimplantation stage of mouse development.Genes to Cells 12/2006; 11(11):1281-93. DOI:10.1111/j.1365-2443.2006.01019.x · 2.86 Impact Factor