Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate

Abstract

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...

A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M

“…e ., HU treatment) [25]. Here, we provide evidence that this mutant is also involved in modulating DNA replication during unchallenged growth, and exhibits unusual replication-dependent DNA damage that is likely linked to an increased frequency of both apoptotic cell death and spontaneous genome rearrangement under this condition.…”

Proteomic alterations in early stage cervical cancer

“…On average 1,700 proteins per sample (1,296–2,189) were identified. Interestingly, levels of members of the Minichromosome Maintenance (MCM) family ( MCM2 , MCM3 , MCM4 , MCM5 , MCM6 , MCM7 ) [ 23 , 24 ] were found to be highly significantly increased in cervical cancer tissue from early and late stage patients as compared to healthy tissue. A volcano plot illustrates the difference in abundance of proteins in the comparison of both early stage and late stage cervical cancer with the healthy epithelium (Figure 1A and 1B ).…”

FORK-seq: replication landscape of theSaccharomyces cerevisiaegenome by nanopore sequencing

“…For randomized elements, the randomization was repeated 1000 times and the median and [0.01,0.99] percentile interval are shown (red bar). e-f. ECDF of the distance between sporadic (red), clustered (green) and randomly shuffled (black) initiation events, and the center of the closest ORC [25] (e) or Mcm2-7 [27] (f) binding site.…”