DNA Damage Checkpoint Control of Mitosis in Fission Yeast

AbstractStudies of genome stability have exploited visualization of fluorescently tagged proteins in live cells to characterize DNA damage, checkpoint, and repair responses. In this report, we describe a new tool for fission yeast, a tagged version of the end-binding protein Pku70 which is part of the KU protein complex. We compare Pku70 localization to other markers upon treatment to various genotoxins, and identify a unique pattern of distribution. Pku70 provides a new tool to define and characterize DNA lesions and the repair response.

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Histone H3 Lysine 14 Acetylation Is Required for Activation of a DNA Damage Checkpoint in Fission Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m111.329417 ◽

2011 ◽

Vol 287 (6) ◽

pp. 4386-4393 ◽

Cited By ~ 48

Author(s):

Yu Wang ◽

Scott P. Kallgren ◽

Bharat D. Reddy ◽

Karen Kuntz ◽

Luis López-Maury ◽

...

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Histone H3 ◽

Dna Damage Checkpoint

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The Fission Yeast Crb2/Chk1 Pathway Coordinates the DNA Damage and Spindle Checkpoint in Response to Replication Stress Induced by Topoisomerase I Inhibitor

Molecular and Cellular Biology ◽

10.1128/mcb.25.17.7889-7899.2005 ◽

2005 ◽

Vol 25 (17) ◽

pp. 7889-7899 ◽

Cited By ~ 24

Author(s):

Ada Collura ◽

Joel Blaisonneau ◽

Giuseppe Baldacci ◽

Stefania Francesconi

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Topoisomerase I ◽

Genome Stability ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Genetic Material ◽

Spindle Checkpoint ◽

Dna Damage Checkpoint ◽

Replicative Stress

ABSTRACT Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G2/M DNA damage checkpoint.

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The Fission Yeast Rad32 (Mre11)-Rad50-Nbs1 Complex Is Required for the S-Phase DNA Damage Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.23.18.6564-6573.2003 ◽

2003 ◽

Vol 23 (18) ◽

pp. 6564-6573 ◽

Cited By ~ 53

Author(s):

Charly Chahwan ◽

Toru M. Nakamura ◽

Sasirekha Sivakumar ◽

Paul Russell ◽

Nicholas Rhind

Keyword(s):

Dna Damage ◽

Fission Yeast ◽

Chromosome Instability ◽

S Phase ◽

Comparative Genomic ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Damage Repair ◽

Genomic Approach ◽

Telomere Regulation

ABSTRACT Mre11, Rad50, and Nbs1 form a conserved heterotrimeric complex that is involved in recombination and DNA damage checkpoints. Mutations in this complex disrupt the S-phase DNA damage checkpoint, the checkpoint which slows replication in response to DNA damage, and cause chromosome instability and cancer in humans. However, how these proteins function and specifically where they act in the checkpoint signaling pathway remain crucial questions. We identified fission yeast Nbs1 by using a comparative genomic approach and showed that the genes for human Nbs1 and fission yeast Nbs1 and that for their budding yeast counterpart, Xrs2, are members of an evolutionarily related but rapidly diverging gene family. Fission yeast Nbs1, Rad32 (the homolog of Mre11), and Rad50 are involved in DNA damage repair, telomere regulation, and the S-phase DNA damage checkpoint. However, they are not required for G2 DNA damage checkpoint. Our results suggest that a complex of Rad32, Rad50, and Nbs1 acts specifically in the S-phase branch of the DNA damage checkpoint and is not involved in general DNA damage recognition or signaling.

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Genetic Interactions between the Escherichia coli umuDC Gene Products and the β Processivity Clamp of the Replicative DNA Polymerase

Journal of Bacteriology ◽

10.1128/jb.183.9.2897-2909.2001 ◽

2001 ◽

Vol 183 (9) ◽

pp. 2897-2909 ◽

Cited By ~ 29

Author(s):

Mark D. Sutton ◽

Mary F. Farrow ◽

Briana M. Burton ◽

Graham C. Walker

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Cold Sensitivity ◽

Dna Damage Checkpoint ◽

Gene Products ◽

Missense Mutations ◽

Dna Polymerase Iii ◽

Checkpoint Control ◽

Cold Sensitive ◽

Growth Phenotype

ABSTRACT The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA–single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD′ (which enables TLS). In addition, even modest overexpression of theumuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD2C. We have previously reported that overexpression of the ɛ proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of ɛ with UmuD2C is important for the DNA damage checkpoint control function of theumuDC gene products. Here, we report that overexpression of the β processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of theumuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD′C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant β proteins possibly deficient for physical interactions with theumuDC gene products, we selected for noveldnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD′C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of β or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaNmissense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eightdnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of theumuDC gene products, suggesting that the interactions between UmuD′ and β are a subset of those between UmuD and β. Taken together, these findings suggest that interaction of β with UmuD2C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD2C with the ɛ and the β subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.

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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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