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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

ABSTRACT: While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.

SUBMITTER: Munoz-Galvan S

PROVIDER: S-EPMC3561951 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Muñoz-Galván Sandra S López-Saavedra Ana A Jackson Stephen P SP Huertas Pablo P Cortés-Ledesma Felipe F Aguilera Andrés A

Nucleic acids research 20121218 3

While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome sy ...[more]

PMID: 23254329

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Project description:BackgroundMammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth. The molecular mechanisms underpinning this response remain unknown. Since chromatin structure or changes in chromatin structure are prime candidate-B-NHEJ-modulators, we study here the role of chromatin hyperacetylation, either by HDAC2 knockdown or treatment with the HDAC inhibitor TSA, on the repair by B-NHEJ of IR-induced DSBs.ResultssiRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well.ConclusionsIn summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth.

Dataset Information

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Publications

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

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