Mrc1 phosphorylation in response to DNA replication stress is required for Mec1 accumulation at the stalled fork

DNA replication stress activates a response pathway that stabilizes stalled forks and promotes the completion of replication. The budding yeast Mec1 sensor kinase, Mrc1 mediator, and Rad53 effector kinase are central to this signal transduction cascade in S phase. We report that Mec1-dependent, Rad53-independent phosphorylation of Mrc1 is required to establish a positive feedback loop that stabilizes Mec1 and the replisome at stalled forks. A structure–function analysis of Mrc1 also uncovered a central region required for proper mediator function and association with replisome components. Together these results reveal new insight into how Mrc1 facilitates checkpoint signal amplification at stalled replication forks.     

Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae

Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.     

A single unbranched S-phase DNA damage and replication fork blockage checkpoint pathway

The eukaryotic intra-S-phase checkpoint, which slows DNA synthesis in response to DNA damage, is poorly understood. Is DNA damage recognized directly, or indirectly through its effects on replication forks? Is the slowing of S phase in part because of competition between DNA synthesis and recombination/repair processes? The results of our genetic analyses of the intra-S-phase checkpoint in the fission yeast, Schizosaccharomyces pombe, suggest that the slowing of S phase depends weakly on the helicases Rqh1 and Srs2 but not on other recombination/repair pathways. The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 (similar to budding yeast Dpb11, which interacts with DNA polymerase epsilon) but not on Rhp9 (similar to budding yeast Rad9, necessary for direct damage recognition). These results suggest that, in fission yeast, the signal activating the intra-S-phase checkpoint is generated only when replication forks encounter DNA damage.     

An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing

The Dbf4Cdc7 kinase acts at the level of individual origins to promote the initiation of DNA replication. We demonstrate through both immunoprecipitation and two-hybrid assays that a domain comprising the first 296 aa of Dbf4p interacts with Orc2p and Orc3p subunits of the origin recognition complex (ORC). Given that the activation of Rad53 kinase in response to the DNA replication checkpoint leads to the release of Dbf4p from an ORC-containing chromatin fraction, we also examined interaction between Dbf4p and Rad53p. This same domain of Dbf4p binds specifically to the forkhead homology-associated (FHA) domains of Rad53p. Cell cycle arrest in G(2)M, provoked by the overexpression of the Dbf4 domain, is suppressed in a rad53 mutant. Moreover, its overexpression perturbs the regulation of late, but not early, origin firing in wild-type cells after treatment with hydroxyurea.     

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. We describe four lines of evidence that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication.     

Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication

Chromosomes are replicated in characteristic, temporal patterns during S phase. We have compared the timing of association of replication proteins at early- and late-replicating origins of replication. Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins. In contrast, Cdc45p association with late origins is delayed relative to early origins. DNA polymerase alpha association is similarly delayed at late origins and requires Cdc45p function. Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins. These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins.     

Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks

The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2–7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.     

The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway

Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct. We have previously shown that the S. cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis.     

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.     

Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts

Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.     

Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5'-3' and the other with 3'-5' polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.     

Suppression of spontaneous genome rearrangements in yeast DNA helicase mutants

Saccharomyces cerevisiae mutants lacking two of the three DNA helicases Sgs1, Srs2, and Rrm3 exhibit slow growth that is suppressed by disrupting homologous recombination. Cells lacking Sgs1 and Rrm3 accumulate gross-chromosomal rearrangements (GCRs) that are suppressed by the DNA damage checkpoint and by homologous recombination-defective mutations. In contrast, rrm3, srs2, and srs2 rrm3 mutants have wild-type GCR rates. GCR types in helicase double mutants include telomere additions, translocations, and broken DNAs healed by a complex process of hairpin-mediated inversion. Spontaneous activation of the Rad53 checkpoint kinase in the rrm3 mutant depends on the Mec3/Rad24 DNA damage sensors and results from activation of the Mec1/Rad9-dependent DNA damage response rather than the Mrc1-dependent replication stress response. Moreover, helicase double mutants accumulate Rad51-dependent Ddc2 foci, indicating the presence of recombination intermediates that are sensed by checkpoints. These findings demonstrate that different nonreplicative helicases function at the interface between replication and repair to maintain genome integrity.     

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.     

Defective p53 engagement after the induction of DNA damage in cells deficient in topoisomerase 3beta

The type IA topoisomerases have been implicated in the repair of dsDNA breaks by homologous recombination and in the resolution of stalled or damaged DNA replication forks; thus, these proteins play important roles in the maintenance of genomic stability. We studied the functions of one of the two mammalian type IA enzymes, Top3beta, using murine embryonic fibroblasts (MEFs) derived from top3beta(-/-) embryos. top3beta(-/-) MEFs proliferated more slowly than TOP3beta(+/+) control MEFs, demonstrated increased sensitivity to DNA-damaging agents such as ionizing and UV radiation, and had increased DNA double-strand breaks as manifested by increased gamma-H2-AX phosphorylation. However, incomplete enforcement of the G(1)-S cell cycle checkpoint was observed in top3beta(-/-) MEFs. Notably, ataxia-telangiectasia, mutated (ATM)/ATM and Rad3-related (ATR)-dependent substrate phosphorylation after UV-B and ionizing radiation was impaired in top3beta(-/-) versus TOP3beta(+/+) control MEFs, and impaired up-regulation of total and Ser-18-phosphorylated p53 was observed in top3beta(-/-) cells. Taken together, these results suggest an unanticipated role for Top3beta beyond DNA repair in the activation of cellular responses to DNA damage.     

Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint

The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad1731) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad1731 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad1731 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad1731 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad1731 showed no detectable exonuclease activity.     

Multiple pathways process stalled replication forks

Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.     

Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases

Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53.     

DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae

In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts. Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1. DRC1 is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins. DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.