Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage

ABSTRACT Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+ is identical to rad35+ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

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Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.1997-2007.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 1997-2007 ◽

Cited By ~ 57

Author(s):

Duncan J. Clarke ◽

Guillaume Mondesert ◽

Marisa Segal ◽

Bonnie L. Bertolaet ◽

Sanne Jensen ◽

...

Keyword(s):

Dna Damage ◽

Spindle Assembly ◽

S Phase ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Checkpoint Control ◽

Checkpoint Signaling ◽

Ubiquitin System ◽

Dependent Cell ◽

S Phase Checkpoint

ABSTRACT In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact withPDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 andDDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

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Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress

Biochemical Pharmacology ◽

10.1016/j.bcp.2006.11.014 ◽

2007 ◽

Vol 73 (6) ◽

pp. 760-772 ◽

Cited By ~ 25

Author(s):

Z. Ping Lin ◽

Michael F. Belcourt ◽

Rocco Carbone ◽

Jana S. Eaton ◽

Philip G. Penketh ◽

...

Keyword(s):

Dna Damage ◽

Ribonucleotide Reductase ◽

Dna Damage Repair ◽

Replication Stress ◽

S Phase ◽

Damage Repair ◽

Ribonucleotide Reductase R2 ◽

S Phase Checkpoint

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Saccharomyces cerevisiae Rrm3p DNA Helicase Promotes Genome Integrity by Preventing Replication Fork Stalling: Viability of rrm3 Cells Requires the Intra-S-Phase Checkpoint and Fork Restart Activities

Molecular and Cellular Biology ◽

10.1128/mcb.24.8.3198-3212.2004 ◽

2004 ◽

Vol 24 (8) ◽

pp. 3198-3212 ◽

Cited By ~ 97

Author(s):

Jorge Z. Torres ◽

Sandra L. Schnakenberg ◽

Virginia A. Zakian

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Replication Fork ◽

Opportunity To Learn ◽

Normal Growth ◽

Dna Helicase ◽

S Phase ◽

Exogenous Dna ◽

Fork Stalling ◽

S Phase Checkpoint

ABSTRACT Rrm3p is a 5′-to-3′ DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.

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Divergent S Phase Checkpoint Activation Arising from Prereplicative Complex Deficiency Controls Cell Survival

Molecular Biology of the Cell ◽

10.1091/mbc.e09-01-0022 ◽

2009 ◽

Vol 20 (17) ◽

pp. 3953-3964 ◽

Cited By ~ 9

Author(s):

Eric Lau ◽

Gary G. Chiang ◽

Robert T. Abraham ◽

Wei Jiang

Keyword(s):

Dna Replication ◽

Cancer Cells ◽

S Phase ◽

Checkpoint Control ◽

Multiple Cancer ◽

Checkpoint Activation ◽

Diploid Cells ◽

Prereplicative Complex ◽

Stress Damage ◽

S Phase Checkpoint

The DNA replication machinery plays additional roles in S phase checkpoint control, although the identities of the replication proteins involved in checkpoint activation remain elusive. Here, we report that depletion of the prereplicative complex (pre-RC) protein Cdc6 causes human nontransformed diploid cells to arrest nonlethally in G1-G1/S and S phase, whereas multiple cancer cell lines undergo G1-G1/S arrest and cell death. These divergent phenotypes are dependent on the activation, or lack thereof, of an ataxia telangiectasia and Rad3-related (ATR)-dependent S phase checkpoint that inhibits replication fork progression. Although pre-RC deficiency induces chromatin structural alterations in both nontransformed and cancer cells that normally lead to ATR checkpoint activation, the sensor mechanisms in cancer cells seem to be compromised such that higher levels of DNA replication stress/damage are required to trigger checkpoint response. Our results suggest that therapy-induced disruption of pre-RC function might exert selective cytotoxic effects on tumor cells in human patients.

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Critical Role for Mouse Hus1 in an S-Phase DNA Damage Cell Cycle Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.23.3.791-803.2003 ◽

2003 ◽

Vol 23 (3) ◽

pp. 791-803 ◽

Cited By ~ 50

Author(s):

Robert S. Weiss ◽

Philip Leder ◽

Cyrus Vaziri

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Synthesis ◽

Critical Role ◽

S Phase ◽

Cell Cycle Checkpoint ◽

Dna Damage Checkpoint ◽

Null Mouse ◽

Cycle Checkpoint ◽

S Phase Checkpoint

ABSTRACT Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1− fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G2/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

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A Role for Saccharomyces cerevisiae Chk1p in the Response to Replication Blocks

Molecular Biology of the Cell ◽

10.1091/mbc.e03-11-0792 ◽

2004 ◽

Vol 15 (9) ◽

pp. 4051-4063 ◽

Cited By ~ 18

Author(s):

Kaila L. Schollaert ◽

Julie M. Poisson ◽

Jennifer S. Searle ◽

Jennifer A. Schwanekamp ◽

Craig R. Tomlinson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Transcriptional Response ◽

S Phase ◽

Yeast Cells ◽

Dna Damage Checkpoint ◽

Checkpoint Kinases ◽

Checkpoint Pathway ◽

Dna Replication And Repair ◽

S Phase Checkpoint

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Δ cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Δ dun1Δ cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

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Defective S Phase Chromatin Assembly Causes DNA Damage, Activation of the S Phase Checkpoint, and S Phase Arrest

Molecular Cell ◽

10.1016/s1097-2765(03)00037-6 ◽

2003 ◽

Vol 11 (2) ◽

pp. 341-351 ◽

Cited By ~ 166

Author(s):

Xiaofen Ye ◽

Alexa A Franco ◽

Hidelita Santos ◽

David M Nelson ◽

Paul D Kaufman ◽

...

Keyword(s):

Dna Damage ◽

S Phase ◽

Chromatin Assembly ◽

Phase Arrest ◽

S Phase Arrest ◽

S Phase Checkpoint

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Topoisomerase III Acts Upstream of Rad53p in the S-Phase DNA Damage Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.21.21.7150-7162.2001 ◽

2001 ◽

Vol 21 (21) ◽

pp. 7150-7162 ◽

Cited By ~ 44

Author(s):

Ronjon K. Chakraverty ◽

Jonathan M. Kearsey ◽

Thomas J. Oakley ◽

Muriel Grenon ◽

Maria-Angeles de la Torre Ruiz ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cellular Response ◽

Cell Cycle Progression ◽

Uv Light ◽

S Phase ◽

Dna Damaging Agents ◽

Topoisomerase Iii ◽

Rad53 Kinase ◽

S Phase Checkpoint

ABSTRACT Deletion of the Saccharomyces cerevisiae TOP3gene, encoding Top3p, leads to a slow-growth phenotype characterized by an accumulation of cells with a late S/G2content of DNA (S. Gangloff, J. P. McDonald, C. Bendixen, L. Arthur, and R. Rothstein, Mol. Cell. Biol. 14:8391–8398, 1994). We have investigated the function of TOP3 during cell cycle progression and the molecular basis for the cell cycle delay seen in top3Δ strains. We show that top3Δ mutants exhibit a RAD24-dependent delay in the G2 phase, suggesting a possible role for Top3p in the resolution of abnormal DNA structures or DNA damage arising during S phase. Consistent with this notion,top3Δ strains are sensitive to killing by a variety of DNA-damaging agents, including UV light and the alkylating agent methyl methanesulfonate, and are partially defective in the intra-S-phase checkpoint that slows the rate of S-phase progression following exposure to DNA-damaging agents. This S-phase checkpoint defect is associated with a defect in phosphorylation of Rad53p, indicating that, in the absence of Top3p, the efficiency of sensing the existence of DNA damage or signaling to the Rad53 kinase is impaired. Consistent with a role for Top3p specifically during S phase, top3Δ mutants are sensitive to the replication inhibitor hydroxyurea, expression of the TOP3 mRNA is activated in late G1 phase, and DNA damage checkpoints operating outside of S phase are unaffected by deletion of TOP3. All of these phenotypic consequences of loss of Top3p function are at least partially suppressed by deletion of SGS1, the yeast homologue of the human Bloom's and Werner's syndrome genes. These data implicate Top3p and, by inference, Sgs1p in an S-phase-specific role in the cellular response to DNA damage. A model proposing a role for these proteins in S phase is presented.

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S-Phase Checkpoint Pathways Stimulate the Mobility of the Retrovirus-Like Transposon Ty1

Molecular and Cellular Biology ◽

10.1128/mcb.01095-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8874-8885 ◽

Cited By ~ 29

Author(s):

M. Joan Curcio ◽

Alison E. Kenny ◽

Sharon Moore ◽

David J. Garfinkel ◽

Matthew Weintraub ◽

...

Keyword(s):

Dna Damage ◽

Reverse Transcriptase ◽

Reverse Transcriptase Activity ◽

S Phase ◽

Dna Lesions ◽

Restriction Factors ◽

Transcriptase Activity ◽

Checkpoint Pathway ◽

Ty1 Mobility ◽

S Phase Checkpoint

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

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