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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    • "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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    • "b The leading strand passes through the central channel of the CMG, while the lagging strand makes strong contacts with a ssDNA binding groove of Cdc45 and minor contacts with MCM2–7 on its outer C-terminal surface and GINS. The EM map of the CMG complex is based on EMDB-1832 (Costa et al. 2011) Chromosoma Cdc45 only bind to origins upon DDK activation (Heller et al. 2011; Natsume et al. 2013; Tanaka et al. 2011). To establish temporal control of DNA replication the activity of DDK must be regulated. "
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    ABSTRACT: A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2-7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2-7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.
    Chromosoma 10/2014; 124(1). DOI:10.1007/s00412-014-0489-2 · 4.60 Impact Factor
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    • "The decreased density of ORC will result in fewer potential replication origins in the late replicating domains. Activation of replication origins is regulated in part by CDK and DDK activities during S phase (Heller et al. 2011). These CDK substrates and DDK activity are limiting for origin activation during S phase and their overexpression can promote the early activation of normally late replicating origins in S. cerevisiae (Mantiero et al. 2011; Tanaka et al. 2011). "
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    ABSTRACT: DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.
    Genome Research 07/2014; 24(7). DOI:10.1101/gr.160010.113 · 14.63 Impact Factor
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