“…This activity specifically requires the Mcm6/2 ATPase active site that was previously implicated as having a regulatory rather than a DNA-unwinding role in Mcm2-7 (32,68,69). Correspondingly, reference mcm alleles that do not affect the Mcm6/2 active site have apparently normal checkpoint signaling (Fig.…”
Section: Discussionmentioning
confidence: 80%
“…The Mcm6/2 active site biochemically functions to regulate rather than directly participate in DNA unwinding. Early work showed that a specific Mcm subcomplex (Mcm467) was competent to unwind DNA (71), while the addition of the remaining Mcm subunits (i.e., Mcm2, -3, and -5) inhibited this activity (69,72). In part, this inhibition depends upon conserved ATPase motifs in Mcm2 (i.e., the function of the Mcm6/2 active site) rather than altered oligomerization caused by Mcm2 binding to the Mcm467 subcomplex (69); mutations that reduce but do not eliminate ATP hydrolysis at this site are viable but demonstrate sensitivity to DNA-damaging agents, consistent with a potential checkpoint defect (68).…”
The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.
The eukaryotic DNA replication checkpoint (DRC) monitors chromosome duplication during S phase and, if irregularities occur, facilitates numerous cellular responses that promote genome stability. In budding yeast, this checkpoint depends upon a phosphorylation cascade initiated by the upstream sensor kinase Mec1/ATR (1, 2), which in turn leads to the phosphorylation and activation of the Mrc1/claspin mediator (3, 4) and ultimately the effector kinase Rad53/Chk1 (5, 6). During deoxynucleoside triphosphate (dNTP) limitation or other forms of replication stress, DRC activation protects genome stability by Rad53-dependent phosphorylation of multiple downstream targets that serve to stabilize nascent replication forks and blocks cell cycle progression, inappropriate recombination (7-9), and the activation of late origins until the stress is alleviated (reviewed in reference 10). In addition to the DRC, a second related pathway that specifically monitors and responds to DNA damage and double-strand breaks also operates during S phase (DNA damage checkpoint [DDC]) (reviewed in reference 11).How the DRC cascade mechanistically interacts with the core replication machinery is incompletely understood. Current evidence indicates that replication plays a passive role in the process. DNA lesions or stress causes a physical uncoupling between DNA polymerase and the replicative helicase; this in turn results in an aberrantly increased level of single-stranded DNA (ssDNA) production that leads to checkpoint activation (12-14). Correspondingly, normal replication fork formation is a prerequisite for DRC activation (15-18).However, strong interactions between DRC components and core replication factors, even in the absence of replication stress, suggest that DNA replication in general and the MCM replicative helicase in particular play broader roles in the DRC. The mediator proteins in the cascade (Mrc1/claspin, Tof1/Timeless, and Csm3/ Tipin) physically interact with and stabilize both Mcm2-7 and DNA polymerase ε (19-23) and protect fork integrity during replication stress (21,2...
“…This activity specifically requires the Mcm6/2 ATPase active site that was previously implicated as having a regulatory rather than a DNA-unwinding role in Mcm2-7 (32,68,69). Correspondingly, reference mcm alleles that do not affect the Mcm6/2 active site have apparently normal checkpoint signaling (Fig.…”
Section: Discussionmentioning
confidence: 80%
“…The Mcm6/2 active site biochemically functions to regulate rather than directly participate in DNA unwinding. Early work showed that a specific Mcm subcomplex (Mcm467) was competent to unwind DNA (71), while the addition of the remaining Mcm subunits (i.e., Mcm2, -3, and -5) inhibited this activity (69,72). In part, this inhibition depends upon conserved ATPase motifs in Mcm2 (i.e., the function of the Mcm6/2 active site) rather than altered oligomerization caused by Mcm2 binding to the Mcm467 subcomplex (69); mutations that reduce but do not eliminate ATP hydrolysis at this site are viable but demonstrate sensitivity to DNA-damaging agents, consistent with a potential checkpoint defect (68).…”
The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.
The eukaryotic DNA replication checkpoint (DRC) monitors chromosome duplication during S phase and, if irregularities occur, facilitates numerous cellular responses that promote genome stability. In budding yeast, this checkpoint depends upon a phosphorylation cascade initiated by the upstream sensor kinase Mec1/ATR (1, 2), which in turn leads to the phosphorylation and activation of the Mrc1/claspin mediator (3, 4) and ultimately the effector kinase Rad53/Chk1 (5, 6). During deoxynucleoside triphosphate (dNTP) limitation or other forms of replication stress, DRC activation protects genome stability by Rad53-dependent phosphorylation of multiple downstream targets that serve to stabilize nascent replication forks and blocks cell cycle progression, inappropriate recombination (7-9), and the activation of late origins until the stress is alleviated (reviewed in reference 10). In addition to the DRC, a second related pathway that specifically monitors and responds to DNA damage and double-strand breaks also operates during S phase (DNA damage checkpoint [DDC]) (reviewed in reference 11).How the DRC cascade mechanistically interacts with the core replication machinery is incompletely understood. Current evidence indicates that replication plays a passive role in the process. DNA lesions or stress causes a physical uncoupling between DNA polymerase and the replicative helicase; this in turn results in an aberrantly increased level of single-stranded DNA (ssDNA) production that leads to checkpoint activation (12-14). Correspondingly, normal replication fork formation is a prerequisite for DRC activation (15-18).However, strong interactions between DRC components and core replication factors, even in the absence of replication stress, suggest that DNA replication in general and the MCM replicative helicase in particular play broader roles in the DRC. The mediator proteins in the cascade (Mrc1/claspin, Tof1/Timeless, and Csm3/ Tipin) physically interact with and stabilize both Mcm2-7 and DNA polymerase ε (19-23) and protect fork integrity during replication stress (21,2...
“…Figure 6.ssDNA binding by Mcm2–7. ( A ) DNA binding was measured using a gel filtration-based assay (6). Binding of Mcm2 WT –7 (open triangle), Mcm2 EE –7 (filled square) and Mcm2 KR –7 (open circle) was determined in triplicate experiments and the mean plotted.…”
The S-phase kinase, DDK controls DNA replication through phosphorylation of the replicative helicase, Mcm2–7. We show that phosphorylation of Mcm2 at S164 and S170 is not essential for viability. However, the relevance of Mcm2 phosphorylation is demonstrated by the sensitivity of a strain containing alanine at these positions (mcm2AA) to methyl methanesulfonate (MMS) and caffeine. Consistent with a role for Mcm2 phosphorylation in response to DNA damage, the mcm2AA strain accumulates more RPA foci than wild type. An allele with the phosphomimetic mutations S164E and S170E (mcm2EE) suppresses the MMS and caffeine sensitivity caused by deficiencies in DDK function. In vitro, phosphorylation of Mcm2 or Mcm2EE reduces the helicase activity of Mcm2–7 while increasing DNA binding. The reduced helicase activity likely results from the increased DNA binding since relaxing DNA binding with salt restores helicase activity. The finding that the ATP site mutant mcm2K549R has higher DNA binding and less ATPase than mcm2EE, but like mcm2AA results in drug sensitivity, supports a model whereby a specific range of Mcm2–7 activity is required in response to MMS and caffeine. We propose that phosphorylation of Mcm2 fine-tunes the activity of Mcm2–7, which in turn modulates DNA replication in response to DNA damage.
“…These observations have led to the suggestion that Mcm2, 3 and 5 may play essential regulatory roles within Mcm2-7. Recent mutational studies are consistent with that idea [11,46]. However, the proposal that only a subset of Mcm2-7 subunits participates directly in DNA unwinding must be reconciled with studies on homohexameric helicases in which all subunits participate directly in the molecular mechanisms required for DNA translocation and DNA unwinding [6,56].…”
BackgroundMinichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7.ResultsHere, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer.ConclusionsThe minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.
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