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Homologous recombination and Mus81 promote replication completion in response to replication fork blockage

ABSTRACT: Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway has been involved in the restart of blocked replication forks. Here, we used a method to increase yeast cell permeability in order to study at the molecular level the fate of replication forks blocked by DNA topoisomerase I poisoning by camptothecin (CPT). Our results indicate that Rad52 and Rad51 HR factors are required to complete DNA replication in response to CPT. Recombination events occurring during S phase do not generally lead to the restart of DNA synthesis but rather protect blocked forks until they merge with convergent forks. This fusion generates structures requiring their resolution by the Mus81 endonuclease in G2/M. At the global genome level, the multiplicity of replication origins in eukaryotic genomes and the fork protection mechanism provided by HR appear therefore to be essential to complete DNA replication in response to fork blockage.

SUBMITTER: Dr. Benjamin Pardo

PROVIDER: S-SCDT-EMBOR-2019-49367V1 | biostudies-other |

REPOSITORIES: biostudies-other

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Homologous recombination and Mus81 promote replication completion in response to replication fork blockage.

Project description:Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway has been involved in the restart of blocked replication forks. Here, we used a method to increase yeast cell permeability in order to study at the molecular level the fate of replication forks blocked by DNA topoisomerase I poisoning by camptothecin (CPT). Our results indicate that Rad52 and Rad51 HR factors are required to complete DNA replication in response to CPT. Recombination events occurring during S phase do not generally lead to the restart of DNA synthesis but rather protect blocked forks until they merge with convergent forks. This fusion generates structures requiring their resolution by the Mus81 endonuclease in G2 /M. At the global genome level, the multiplicity of replication origins in eukaryotic genomes and the fork protection mechanism provided by HR appear therefore to be essential to complete DNA replication in response to fork blockage.

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Nek1 Regulates Rad54 to Orchestrate Homologous Recombination and Replication Fork Stability.

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Acute MUS81 depletion leads to replication fork slowing and a constitutive DNA damage response.

Project description:The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity.

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PARP1 inhibition radiosensitizes HNSCC cells deficient in homologous recombination by disabling the DNA replication fork elongation response.

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