Eukaryotic Origin-Dependent DNA Replication In Vitro Reveals Sequential Action of DDK and S-CDK Kinases

Howard Hughes Medical Institute, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Cell (Impact Factor: 32.24). 07/2011; 146(1):80-91. DOI: 10.1016/j.cell.2011.06.012
Source: PubMed


Proper eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) but not S phase cyclin-dependent kinase (S-CDK) is required for the initial origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Likewise, in vivo, DDK drives early-firing-origin recruitment of Cdc45 before activation of S-CDK. After S-CDK activation, a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Finally, recruitment of lagging but not leading strand DNA polymerases depends on Mcm10 and DNA unwinding. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to origin DNA unwinding and RNA primer synthesis.

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    • "Sites of initiation are specified by the pre-replication complex (pre-RC), which includes ORC, CDC6 and CDT1, leading to recruitment of the replicative helicase complex containing MCM2–7, CDC45 and GINS (CMG complex) (Ilves et al., 2010; Pacek et al., 2006). DDK activates the CMG complex leading to localised DNA unwinding (Heller et al., 2011), and in yeast this is followed by CDK-mediated phosphorylation of GINS subunits SLD2 and SLD3, recruitment of DPB11 and polymerase e to form the pre-initiation complex (pre- IC), and activation of MCM helicase activity (Heller et al., 2011; Muramatsu et al., 2010; Tanaka et al., 2007; Zegerman and Diffley, 2007). Subsequent recruitment of PCNA and polymerases a and d complete the replisome and enable initiation of DNA replication. "
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    ABSTRACT: CIZ1 is a nuclear matrix protein that cooperates with cyclin A/CDK2 to promote mammalian DNA replication. We show here that cyclin A/CDK2 also negatively regulates CIZ1 activity via phosphorylation at threonines 144, 192, and 293. Phosphomimetic mutants do not promote DNA replication in cell-free and cell-based assays, and also have a dominant negative effect on replisome formation at the level of PCNA recruitment. Phosphorylation blocks direct interaction with cyclin A/CDK2, and recruitment of endogenous cyclin A to the nuclear matrix. In contrast, phosphomimetic CIZ1 retains nuclear matrix binding capability, and interaction with CDC6 is not affected. Phospho-threonine 192-specific antibodies confirm that CIZ1 is phosphorylated during S-phase and G2, and show that phosphorylation at this site occurs at post-initiation concentrations of cyclin A/CDK2. Together the data suggest that CIZ1 is a kinase sensor that promotes initiation of DNA replication at low kinase levels, when in a hypophosphorylated state that is permissive for cyclin A-CDK2 interaction and delivery to licensed origins, but blocks delivery at higher kinase levels when it is itself phosphorylated.
    Journal of Cell Science 03/2015; 128(8). DOI:10.1242/jcs.161919 · 5.43 Impact Factor
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    • "Although the most direct interpretation of our data supports only one Mcm2–7 double hexamer per origin, we cannot rule out more complex models of Mcm2–7 loading (including transient and weak interactions ) that may result in multiple complexes per origin. Elegant in vitro experiments reconstituting pre-RC assembly (Speck et al. 2005; Fern andez-Cid et al. 2013; Frigola et al. 2013; Sun et al. 2013) and replication initiation (Remus et al. 2009; Heller et al. 2011) have provided tremendous mechanistic insights, but assays performed on template DNA clearly lack the regulatory complexity observed at each origin in its chromosomal complex. For example, unlike the situation in vivo, pre- RC assembly and initiation on template DNA are not strictly dependent on origin DNA sequences (Remus et al. 2009; Gros et al. 2014). "
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    ABSTRACT: Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). © 2015 Belsky et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 01/2015; 29(2):212-224. DOI:10.1101/gad.247924.114. · 10.80 Impact Factor
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    • "The Mcm2–7 double-hexamer architecture provides a structural basis for it being a DDK target DDK acts on the Mcm2–7 double hexamer but not the single Mcm2–7 hexamer in solution and in vivo (Sheu and Stillman 2006; Randell et al. 2010; Heller et al. 2011; Ramer et al. 2013; Tanaka and Araki 2013), and the DDK action precedes the S-CDK action to activate the steps toward the actual initiation of DNA synthesis at each origin (Heller et al. 2011). However, it was unclear how DDK distinguishes a double hexamer from the single hexamer, as they both contain the same protein components . "
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    ABSTRACT: Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
    Genes & Development 10/2014; 28(20):2291-303. DOI:10.1101/gad.242313.114 · 10.80 Impact Factor
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