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.     

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.     

Induction of a G1-S checkpoint in fission yeast

Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.     

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.     

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.     

Activation of an ataxia telangiectasia mutation-dependent intra-S-phase checkpoint by anti-tumour drugs in HL-60 and human lymphoblastoid cells

In yeast cells, the intra-S-phase checkpoint slows down the rate of DNA replication in response to DNA damage. Here we showed that a similar checkpoint mechanism is present and activated by anti-tumour drugs in HL-60 and Epstein-Barr virus (EBV)-transformed human lymphoblastoid cells. Using bromodeoxyuridine (BrdU) pulse labelling combined with two-dimensional flow cytometric analysis, we clearly visualized the cell-cycle progression of the BrdU-positive population (cells originally belonging to the S phase) and detected even subtle changes in S-phase progression induced by mild drug treatment conditions free of apoptosis. The DNA topoisomerase II inhibitors, doxorubicin and etoposide (250 nmol/l and 400 nmol/l, respectively, for 8 h), retained the BrdU-positive HL-60 cells in the latter half of S and G2/M positions, and the pyrimidine analogue anti-metabolite, cytosine beta-D-arabinofuranose (Ara-C; 50 nmol/l), kept them in early-to-late S phase after 8 h of incubation. Because 10 micromol/l of caffeine added 2 h later attenuated the S-phase retardation by these drugs in HL-60 cells, slowing of the S-phase progression should be actively regulated. Furthermore, two ataxia telangiectasia (AT)-derived lymphoblastoid cell lines were impaired in the doxorubicin-induced S-phase retardation, which indicated that the process is at least partially dependent on ataxia telangiectasia mutated (ATM) gene product. The inhibitory mechanism on S-phase progression elicited by anti-tumour drugs in HL-60 and lymphoblastoid cells may therefore correspond to the intra-S-phase checkpoint of the yeast cells.     

Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.     

Postreplication gaps at UV lesions are signals for checkpoint activation

Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polκ and Polη significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polζ repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polκ and Polη is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.     

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by gamma-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.     

Polymerase eta deficiency in the xeroderma pigmentosum variant uncovers an overlap between the S phase checkpoint and double-strand break repair

The xeroderma pigmentosum variant (XPV) is a genetic disease involving high levels of solar-induced cancer that has normal excision repair but shows defective DNA replication after UV irradiation because of mutations in the damage-specific polymerase hRAD30. We previously found that the induction of sister chromatid exchanges by UV irradiation was greatly enhanced in transformed XPV cells, indicating the activation of a recombination pathway. We now have identified that XPV cells make use of a homologous recombination pathway involving the hMre11/hRad50/Nbs1 protein complex, but not the Rad51 recombination pathway. The hMre11 complexes form at arrested replication forks, in association with proliferating cell nuclear antigen. In x-ray-damaged cells, in contrast, there is no association between hMre11 and proliferating cell nuclear antigen. This recombination pathway assumes greater importance in transformed XPV cells that lack a functional p53 pathway and can be detected at lower frequencies in excision-defective XPA fibroblasts and normal cells. DNA replication arrest after UV damage, and the associated S phase checkpoint, is therefore a complex process that can recruit a recombination pathway that has a primary role in repair of double-strand breaks from x-rays. The symptoms of elevated solar carcinogenesis in XPV patients therefore may be associated with increased genomic rearrangements that result from double-strand breakage and rejoining in cells of the skin in which p53 is inactivated by UV-induced mutations.     

Schizosaccharomyces pombe cdc20+ encodes DNA polymerase ɛ and is required for chromosomal replication but not for the S phase checkpoint

In fission yeast both DNA polymerase alpha (pol α) and delta (pol δ) are required for DNA chromosomal replication. Here we demonstrate that Schizosaccharomyces pombe cdc20⁺ encodes the catalytic subunit of DNA polymerase epsilon (pol ) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature, cdc20 temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that cdc20⁺ is required early in S phase very near to the initiation step. In the budding yeast Saccharomyces cerevisiae, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995) Cell 80, 29–39]. We have investigated whether cdc20⁺ is required for the checkpoint control operating in fission yeast, and our data indicate that pol does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol α rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol α, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.     

Intra-G1 arrest in response to UV irradiation in fission yeast

G1 is a crucial phase of cell growth because the decision to begin another mitotic cycle is made during this period. Occurrence of DNA damage in G1 poses a particular challenge, because replication of damaged DNA can be deleterious and because no sister chromatid is present to provide a template for recombinational repair. We therefore have studied the response of Schizosaccharomyces pombe cells to UV irradiation in early G1 phase. We find that irradiation results in delayed progression through G1, as manifested most critically in the delayed formation of the pre-replication complex. This delay does not have the molecular hallmarks of known checkpoint responses: it is independent of the checkpoint proteins Rad3, Cds1, and Chk1 and does not elicit inhibitory phosphorylation of Cdc2. Irradiated cells eventually progress into S phase and arrest in early S by a rad3- and cds1-dependent mechanism, most likely the intra-S checkpoint. Caffeine alleviates both the intra-G1- and intra-S-phase delays. We suggest that intra-G1 delay may be widely conserved and discuss significance and possible mechanisms.     

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.     

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.     

Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast

Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage. This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosphorylated in response to DNA damage. Here we report that Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3.     

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.     

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.     

Frag1, a homolog of alternative replication factor C subunits, links replication stress surveillance with apoptosis

We report the identification and characterization of a potent regulator of genomic integrity, mouse and human FRAG1 gene, a conserved homolog of replication factor C large subunit that is homologous to the alternative replication factor C subunits Elg1, Ctf18/Chl12, and Rad24 of budding yeast. FRAG1 was identified in a search for key caretaker genes involved in the regulation of genomic stability under conditions of replicative stress. In response to stress, Atr participates in the down-regulation of FRAG1 expression, leading to the induction of apoptosis through the release of Rad9 from damaged chromatin during the S phase of the cell cycle, allowing Rad9-Bcl2 association and induction of proapoptotic Bax protein. We propose that the Frag1 signal pathway, by linking replication stress surveillance with apoptosis induction, plays a central role in determining whether DNA damage is compatible with cell survival or whether it requires cell elimination by apoptosis.