Article

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

ABSTRACT

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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    • "To date, ∼80 direct targets of Mec1 have been identified. They include components of the RPA complex (Rfa1 and Rfa2; Smolka et al., 2007), the GINS complex (Psf1; De Piccoli et al., 2012), and the MCM-helicase complex (Mcm4 and Mcm6; Randell et al., 2010), all of which are directly involved in DNA replication. Therefore, inactivation of Mec1 might result in a system-wide failure in genome duplication stemming from inability to phosphorylate key components of the replication machinery. "
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    ABSTRACT: Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.
    Preview · Article · Nov 2015 · Biology Open
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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.
    Full-text · Article · Sep 2015 · Molecular cell
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    • "Although cells that lack Pol32 are viable, they experience replication stress that leads to phosphorylation of the checkpoint kinase Rad53, S phase delay, accumulation in G2/M, and hypersensitivity to DNA damaging agents [24] [27] [35]. When replication forks stall due to exposure to the DNA damaging agents hydroxyurea (HU) or methyl methanesulfonate (MMS), which deplete the dNTP pool and generate base adducts in the template strand, respectively, activation of the replication checkpoint helps to maintain the activity of the replisome, possibly through phosphorylation, that permits fork restart after withdrawal of the genotoxin [36] [37] [38]. Although Exo1 has been found at these stalled forks the presence of the intact replisome is thought to prevent it from acting on the nascent strands [26]. "
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    ABSTRACT: Exo1 belongs to the Rad2 family of structure-specific nucleases and possesses 5′–3′ exonuclease activity on double-stranded DNA substrates. Exo1 interacts physically with the DNA mismatch repair (MMR) proteins Msh2 and Mlh1 and is involved in the excision of the mispaired nucleotide. Independent of its role in MMR, Exo1 contributes to long-range resection of DNA double-strand break (DSB) ends to facilitate their repair by homologous recombination (HR), and was recently identified as a component of error-free DNA damage tolerance pathways. Here, we show that Exo1 activity increases the hydroxyurea sensitivity of cells lacking Pol32, a subunit of DNA polymerases δ and ζ. Both, phospho-mimicking and dephospho-mimicking exo1 mutants act as hypermorphs, as evidenced by an increase in HU sensitivity of pol32Δ cells, suggesting that they are trapped in an active form and that phosphorylation of Exo1 at residues S372, S567, S587, S692 is necessary, but insufficient, for the accurate regulation of Exo1 activity at stalled replication forks. In contrast, neither phosphorylation status is important for Exo1's role in MMR or in the suppression of genome instability in cells lacking Sgs1 helicase. This ability of an EXO1 deletion to suppress the HU hypersensitivity of pol32Δ cells is in contrast to the negative genetic interaction between deletions of EXO1 and POL32 in MMS-treated cells as well as the role of EXO1 in DNA-damage treated rad53 and mec1 mutants.
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