Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60

Miyabe, Izumi; Morishita, Takashi; Shinagawa, Hideo; Carr, Antony

doi:10.1242/jcs.046508

Cited by 13 publications

(14 citation statements)

References 43 publications

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“…Previous studies showed that Cds1 limits ssDNA accumulation in part by restraining MCM activity while also blocking Mus81-dependent damage and preventing late origin firing (5,14,24,32). Our data are consistent with this: in cds1⌬ cells, we observe increased RPA accumulation that is MCM dependent, together with Mus81-dependent phospho-H2A.…”

Section: Discussionsupporting

confidence: 92%

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Sabatinos

Green

Forsburg

2012

Molecular and Cellular Biology

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Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1⌬ and mrc1⌬ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1⌬ and mrc1⌬ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block. Fission yeast Schizosaccharomyces pombe cells treated with hydroxyurea (HU) suffer nucleotide depletion and activate the intra-S phase checkpoint. This causes reversible cell cycle arrest in early S phase and prevents firing of late replication origins (12,19,62). The intra-S phase checkpoint stabilizes existing DNA replication forks and promotes recovery and replication resumption upon release. Loss of the intra-S phase checkpoint corrupts replication forks, leading to DNA damage and cell death. However, the events leading to successful recovery or fork collapse remain poorly defined.HU-treated replication forks synthesize 5 to 10 kb of DNA before they slow in budding yeast (4,27,49). Evidence from several model systems suggests that MCM helicase and polymerases are briefly uncoupled during fork slowing, which leads to increased DNA unwinding and accumulation of replication protein A (RPA) on single-stranded DNA (ssDNA) (37,39,42,43). This promotes Rad3 (ATR, or ScMec1) kinase activation (36, 66), which activates Cds1 kinase (ScRad53) (26). Cds1 inhibits the origin-activating Hsk1 kinase (ScCdc7) to block late origin firing (24,51,54), and the Mcm4 subunit of the MCM helicase is a likely Cds1 substrate (5). Cds1 also phosphorylates the Mus81 nuclease, preventing accumulation of toxic structures (7,22). Finally, Cds1 phosphorylates Cdc25, preventing mitosis in the presence of damage (6,26,65).The mediator protein Mrc1 recruits Cds1 to Rad3 to promote efficient checkpoint activation, but Mrc1 is also a nonessential component of the replication fork complex (56,57,63,64). Mrc1 couples the leading-strand DNA polymerase epsilon (Polε) to the MCM helicase (23,28,41) and regulates recombination during replication stalling and restart (1,22,47).The absence of either Cds1/ScRad53 or Rad3/ScMec1 during HU treatment leads to late origin firing, DNA unwinding, and cell death (2,12,26,49,52). This is accompanied by DNA doublestrand breaks (DSBs), histone H2A phosphorylation (phospho-H2A; H2Ax in metazoans), and accumulation of the Rad52 recombination protein in repair foci...

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Section: Discussionsupporting

confidence: 92%

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Sabatinos

Green

Forsburg

2012

Molecular and Cellular Biology

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“…44 Rad60 phosphorylation is also required to regulate HR at stalled replication forks by ensuring the structural maintenance of the protein complex of chromosomes 5 and 6 (Smc5/6). 45 Nrm1, a transcriptional repressor that acts at the G 1 to S phase transition, is phosphorylated by Cds1 in response to DNA replication stress, which leads to the expression of genes important for DNA replication and repair. 46 Cdc25, a well known Cds1 target, is also phosphorylated by Mek1, which also regulates the nuclear localization of Cdc25 and cell cycle arrest.…”

Section: Methodsmentioning

confidence: 99%

The Mek1 phosphorylation cascade plays a role in meiotic recombination ofSchizosaccharomyces pombe

et al. 2010

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“…However, the modest sensitivity caused by cds1Δ indicated a role for the replication checkpoint during treatment. Cds1 is particularly important for replication fork stalling and restart (e.g., Lindsay et al 1998;Kim and Huberman 2001;Miyabe et al 2009). The homologous recombination protein Rad52 forms nuclear foci in response to a variety of lesions, including double-strand breaks and collapsed or restarting replication forks (Meister et al 2005;Bailis et al 2008).…”

Section: Loss Of the Replication Checkpoint Influences Damage Responsementioning

confidence: 99%

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

et al. 2013

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Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-29-deoxyuridine (BrdU) and 5-ethynyl-29-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

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Schizosaccharomyces pombe Cds1Chk2 regulates homologous recombination at stalled replication forks through the phosphorylation of recombination protein Rad60

Cited by 13 publications

References 43 publications

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

The Mek1 phosphorylation cascade plays a role in meiotic recombination ofSchizosaccharomyces pombe

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

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