The Saccharomyces cerevisiae F-Box Protein Dia2 Is a Mediator of S-Phase Checkpoint Recovery from DNA Damage

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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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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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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SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5829-5842.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5829-5842

Author(s):

P Zheng ◽

D S Fay ◽

J Burton ◽

H Xiao ◽

J L Pinkham ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Dna Synthesis ◽

Genomic Library ◽

Excision Repair ◽

Low Frequency ◽

S Phase ◽

Upstream Region ◽

Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

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Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

10.1101/2020.10.26.355008 ◽

2020 ◽

Author(s):

Christophe de La Roche Saint-André ◽

Vincent Géli

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Genetic Material ◽

S Phase ◽

Replication Forks ◽

H3k4 Methylation ◽

Origin Firing ◽

Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

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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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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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A Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5535 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5535-5547 ◽

Cited By ~ 71

Author(s):

Tadayuki Takeda ◽

Keiko Ogino ◽

Etsuko Matsui ◽

Min Kwan Cho ◽

Hiroyuki Kumagai ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Fission Yeast ◽

Kinase Activity ◽

S Phase ◽

Regulatory Subunit ◽

Checkpoint Control ◽

Regulatory Subunits ◽

Hydroxyurea Treatment ◽

S Phase Checkpoint

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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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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