Mec1 Is One of Multiple Kinases that Prime the Mcm2-7 Helicase for Phosphorylation by Cdc7

Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Molecular cell (Impact Factor: 14.02). 11/2010; 40(3):353-63. DOI: 10.1016/j.molcel.2010.10.017
Source: PubMed


Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms that modulate origin firing and replication fork assembly during cell cycle progression.

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    • "The transient accumulation of ssDNA in S - phase cells may trigger limited ATR activation , thereby coordinating RRM2 accumulation and origin firing . Interestingly , the budding yeast ATR homolog Mec1 is required for priming the Mcm2 - 7 helicase for phosphory - lation by Cdc7 ( Randell et al. , 2010 ). The limited ATR activation during S phase may promote origin firing but also restrict it to a tolerable level , preventing ssDNA from accumulating to a high level that triggers replication catastrophe ( see Figure 7A ). "
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    ABSTRACT: The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells.
    Molecular cell 09/2015; 59(6). DOI:10.1016/j.molcel.2015.07.029 · 14.02 Impact Factor
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    • "DDK targets sequences for phosphorylation characterized by an acidic residue at the +1 position, which can be either an acidic amino acid or a negative charge provided by a phosphoserine or phosphothreonine (Charych et al. 2008; Cho et al. 2006; Montagnoli et al. 2006). Indeed, both CDK and the checkpoint kinase Mec1 phosphorylate chromatin-associated MCM2–7, generating priming sites for DDK-dependent phosphorylation , which contributes to efficient helicase activation and genomic stability (Randell et al. 2010). It is likely that DDK has a large number of targets in the cell, but for budding yeast, it was shown that MCM2–7 is the essential target of the kinase, as mutations in subunits of the helicase can bypass the DDK requirement of the cell (Hardy et al. 1997; Sheu and Stillman 2010). "
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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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    • "MCM2-7, within the OCM, might adopt a structure similar to the double-hexamer. To address this question, we used DDK kinase (Cdc7-Dbf4) as a structural sensor, as DDK has been shown to preferentially phosphorylate MCM2-7 within the double-hexamer and less efficiently if pre-RC assembly is blocked by a Cdc6 E224G ATP hydrolysis mutant (23,24). Consistent with previous work, we found that purified MCM2-7 in the absence of other proteins is weakly phosphorylated by DDK on Mcm2, Mcm4 and Mcm6 (Figure 5A, lane 8). "
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    ABSTRACT: The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.
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