Ferreira, M. G. & Cooper, J. P. Two modes of DNA double-strand break repair are reciprocally regulated through fission yeast cell cycle. Genes Dev. 18, 2249-2254

Telomere Biology Laboratory, Cancer Research UK, London WC2A 3PX, UK.
Genes & Development (Impact Factor: 10.8). 10/2004; 18(18):2249-54. DOI: 10.1101/gad.315804
Source: PubMed


Several considerations suggest that levels of the two major modes of double-strand break (DSB) repair, homologous recombination (HR), and nonhomologous end joining (NHEJ), are regulated through the cell cycle. However, this idea has not been explicitly tested. In the absence of the telomere-binding protein Taz1, fission yeast undergo lethal telomere fusions via NHEJ. These fusions occur only during periods of nitrogen starvation and fail to accumulate during logarithmic growth, when the majority of cells are in G2. We show that G1 arrest is the specific nitrogen starvation-induced event that promotes NHEJ between taz1(-) telomeres. Furthermore, the general levels of NHEJ and HR are reciprocally regulated through the cell cycle, so that NHEJ is 10-fold higher in early G1 than in other cell cycle stages; the reverse is true for HR. Whereas NHEJ is known to be dispensable for survival of DSBs in cycling cells, we find that it is critical for repair and survival of DSBs arising during G1.

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    • "Thus, contrary to our prediction , disinhibition of NHEJ by preventing Xlf1 phosphorylation is not sufficient to fuse unprotected chromosome ends in logphase cultures. NHEJ and HR are regulated independently in the fission yeast cell cycle, as inactivation of HR does not lead to increased use of NHEJ in G 2 cells (Ferreira and Cooper, 2004). Conversely, we expected that abnormal activation of NHEJ in G 2 cells will take place in the presence of activated HR, since the disinhibition of NHEJ alone in the xlf1.AA mutant would not affect HR activity. "
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    ABSTRACT: Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2(Cdk1) provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
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    • "In this process, DNA ends are joined without the requirement for a homologous sequence, making NHEJ potentially mutagenic. In contrast, cells that have entered S phase can use the sister chromatid as a template for high-fidelity DSB repair through HR (Aylon et al, 2004; Ferreira & Cooper, 2004; Sonoda et al, 2006). NHEJ and HR are mutually exclusive pathways since DNA-end resection, which generates long stretches of singlestranded DNA (ssDNA), commits cells to HR and prevents repair by NHEJ. "
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    ABSTRACT: Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/CCdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/CCdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/CCdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/CCdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.
    The EMBO Journal 10/2014; 33(23). DOI:10.15252/embj.201489017 · 10.43 Impact Factor
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    • "Despite the importance of cyclin-dependent kinases (CDKs) for the regulation of the DNA damage response (DDR) (1,2,3), it is still enigmatic how CDKs act as activators of DNA repair while being down-regulated by the DNA damage checkpoint. Two possible answers to this puzzle may lie in the temporal or spatial organization of CDKs. "
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    ABSTRACT: Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb253BP1 in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).
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