Replicative stress is a major source of mutations that contribute to chromosome instability and the initiation of cancer (5
). A primary defense against genome instability is S-phase checkpoints, which recognize DNA damage, arrest the cell cycle, and preserve essential replication structures (10
). In response to S-phase agents such as the ribonucleotide reductase inhibitor hydroxyurea (HU), replication forks stall and activate checkpoint kinases (ATR-Chk1 in mammals and Rad3-Cds1 and Mec1-Rad53 in the yeasts Saccharomyces pombe
and Saccharomyces cerevisiae
, respectively) (10
). The primary function of this replication checkpoint is to retain the assembled replisome at stalled forks during arrest: checkpoint defects lead to catastrophic replication fork collapse and cell lethality. However, recent studies have shown that proteins directly involved in DNA replication as well as recombination are required for cells to repair DNA damage, restart replication, and recover from arrest and must interact with the checkpoint to facilitate these functions (10
The minichromosome maintenance (MCM) complex is an essential replicative helicase that consists of six related subunits (MCM2 to MCM7) and is required for the initiation and elongation phases of DNA synthesis (reviewed in reference 22
). The MCM helicase is therefore centrally positioned to monitor and maintain genome stability at the replication fork. In budding yeast, the use of degron alleles of MCM subunits, which degrade the protein upon a shift to high temperature, has demonstrated that MCM function is required for fork progression throughout S phase as well as for synthesis to resume after forks have stalled in HU (36
). In cells lacking the Rad53 protein kinase or its S-phase activator Mrc1, treatment with HU results in excessive DNA unwinding, catastrophic replication fork collapse, and an inability to restart synthesis (12
). Under these conditions, MCM proteins remain chromatin bound, but the leading and lagging strands of the replication fork become uncoupled (12
). In metazoans, MCM subunits are phosphorylated by the ATM/ATR kinases (17
), suggesting that the MCM complex may be a target or an effector of the replication checkpoint.
However, MCM proteins may also promote S-phase genome stability through checkpoint-independent roles (reviewed in references 3
). Although cellular levels of MCM proteins are estimated as 10- to 40-fold excess over the number of replication origins, yeast temperature-sensitive mcm
) that partially reduce MCM protein levels exhibit increased recombination, chromosome loss, and checkpoint sensitivity (32
). In addition, increased expression or amplification of MCM genes is associated with many types of human cancers that are characterized by genomic instability (reviewed in reference 39
) and the mutation or the dysregulation of MCM subunits can induce skin carcinoma and mammary carcinoma in mouse models (30
). These results suggest that the MCM complex plays a central role in protecting genome stability during S phase.
The stabilization of replisome structure and its recovery from replication arrest also depend upon proteins involved in homologous recombination (HR). In budding yeast, the Rad51 strand exchange enzyme and the Sgs1 recombination helicase are required to maintain DNA polymerase
at stalled replication forks (16
). Sgs1 is additionally involved in checkpoint activation (6
). In fission yeast, the SpRad22 (ScRad52) recombination protein is recruited to nuclear foci immediately as cells are released from HU arrest (48
). Although fission yeast rad22
) mutants are proficient for activation of the replication checkpoint, they still show sensitivity to HU (48
). Together, these results suggest that components of the replisome interact with recombination proteins to maintain and restart replication forks.
Previously, we and others have shown that most fission yeast mcm-ts
mutants accumulate approximately 2C DNA content, but undergo a lethal, checkpoint-dependent arrest in late S phase or G2
). In contrast, a degron allele of mcm4ts
blocks cells at or near replication initiation (42
). Here, we use these different mcm
alleles to examine the consequences to cells of inactivating MCM function during S phase in the absence or the presence of forks stalled by HU. We demonstrate that the loss of MCM function generates DNA breaks, cell cycle arrest, and a loss of viability similar to that observed in mutants that undergo replication fork collapse. Consistent with these results, we find that Mcm4 interacts with the checkpoint protein kinase Cds1 and undergoes Cds1-dependent phosphorylation in cells treated with HU. This result suggests that MCM proteins act to maintain replication fork structure both during normal S phase and during S-phase arrest.
Our data additionally suggest that MCM function is required for proper recovery from replication arrest induced by HU. We observed an interaction between the MCM complex and the HR protein Rhp51 (Rad51). Although Rhp51 and other HR proteins are not required to activate the replication checkpoint, to maintain fork structure in HU, or to restart DNA synthesis, we find that the loss of HR function results in chromosome missegregation following release from HU-induced replication arrest (this work; 48
). We suggest that MCM proteins modulate replication fork progression, arrest, and restart and that they couple these functions with the repair of DNA damage to protect S-phase genome stability. Our analysis in fission yeast is complemented by studies of mammalian cells, suggesting that the role of the MCM complex in S-phase genome stability is conserved.