Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

Froget, Benoît; Blaisonneau, Joël; Lambert, Sarah; Baldacci, Giuseppe

doi:10.1091/mbc.e07-07-0728

Cited by 81 publications

(83 citation statements)

References 61 publications

(80 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Although it is not clearly essential for fork collapse in all cell types, the Mus81-Eme1 complex in Schizosaccharomyces pombe plays a crucial role in DSB generation at stalled replication forks (Froget et al 2008), and a similar relationship has been observed in mammalian cells when CHK1 is inhibited (Forment et al 2011). These findings in aggregate suggest that PLK1 could play an active facilitative role in replication fork collapse at the level of both the replisome and cleaving aberrant fork structures.…”

mentioning

confidence: 72%

“…The SLX4-endonuclease complex has previously been implicated as a mediator of replication fork collapse, particularly in cooperation with the MUS81-EME1 complex (Froget et al 2008;Forment et al 2011;Matos et al 2011;Gallo-Fernandez et al 2012;Munoz-Galvan et al 2012;Schwartz et al 2012;Szakal and Branzei 2013). To examine the involvement of this complex in the DSBs generated upon fork stalling in ATR-deficient cells, Crelox conditional SLX4 cells were used to delete Slx4 and determine the effect of its absence on DSB generation following ATR inhibition and fork stalling.…”

Section: Rnf4 and Plk1 Are Required For Slx4-dependent Dsb Formation mentioning

confidence: 99%

See 1 more Smart Citation

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

et al. 2013

View full text Add to dashboard Cite

The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atrdeleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atrdeleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

show abstract

mentioning

confidence: 72%

Section: Rnf4 and Plk1 Are Required For Slx4-dependent Dsb Formation mentioning

confidence: 99%

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

et al. 2013

View full text Add to dashboard Cite

show abstract

“…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 (1,30). These markers are a feature of replication fork collapse (5,14,27), which results in Chk1 kinase activation (26). Because the G 2 -M damage checkpoint is activated, cells do not divide prematurely (8,26,55).…”

mentioning

confidence: 99%

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Sabatinos

Green

Forsburg

2012

Molecular and Cellular Biology

View full text Add to dashboard Cite

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...

show abstract

“…Although CHK1 may play different roles than WEE1 in this process (45), further studies may address whether nucleotide shortage contributes to replication stalling and DNA damage following CHK1 inhibition of reoxygenated cells. Importantly, stalled forks can act as substrates for endonucleases (46), which may cause excessive DNA damage if not controlled properly, and CHK1 may directly or indirectly be involved in suppression of such endonucleases (33,47,48).…”

Section: Discussionmentioning

confidence: 99%

The Efficacy of CHK1 Inhibitors Is Not Altered by Hypoxia, but Is Enhanced after Reoxygenation

Hasvold¹,

Tkacz-Stachowska²,

Rofstad³

et al. 2013

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

Inhibitors of CHK1 are in clinical trials for cancer treatment in combination with DNA-damaging agents. Importantly, it was previously suggested that hypoxic cancer cells may be particularly sensitive to CHK1 inhibition. However, this suggestion was based on studies in severe, toxic levels of hypoxia (anoxia). The influence of less severe hypoxia on the efficacy of CHK1 inhibitors, administered either as single agents or in combination with other treatments, remains to be investigated. Here, we have assayed the effects of the CHK1 inhibitors, AZD7762 and UCN-01, during various hypoxic conditions and after reoxygenation in the absence and presence of ionizing radiation. Treatment with CHK1 inhibitors during acute or prolonged hypoxia (< 0.03%, 0.2%, and 1% O 2 ; 3 h or 20-24 h) gave similar effects on cell survival as treatment with these inhibitors during normoxia. CHK1 inhibitors combined with ionizing radiation showed similar radiosensitization in hypoxic and normoxic cells. However, when the inhibitors were administered after reoxygenation following prolonged hypoxia (< 0.03% and 0.2%; 20-24 h), we observed decreased cell survival and stronger induction of the DNA damage marker, gH2AX, in S-phase cells. This was accompanied by enhanced phosphorylation of the single-stranded DNA-binding replication protein A. These results suggest that the cytotoxic effects of CHK1 inhibitors are enhanced after reoxygenation following prolonged hypoxia, most likely due to the increased replication-associated DNA damage. Combining CHK1 inhibitors with other treatments that cause increased reoxygenation, such as fractionated radiotherapy, might therefore be beneficial. Mol Cancer Ther; 12(5); 705-16. Ó2013 AACR.

show abstract

Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

Cited by 81 publications

References 61 publications

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

The Efficacy of CHK1 Inhibitors Is Not Altered by Hypoxia, but Is Enhanced after Reoxygenation

Contact Info

Product

Resources

About