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Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation.

ABSTRACT: Rad51 proteins share both structural and functional homologies with the bacterial recombinase RecA. The human Rad51 (HsRad51) is able to catalyse strand exchange between homologous DNA molecules in vitro . However the biological functions of Rad51 in mammals are largely unknown. In order to address this question, we have cloned hamster Rad51 cDNA and overexpressed the corresponding protein in CHO cells. We found that 2-3-fold overexpression of the protein stimulated the homologous recombination between integrated genes by 20-fold indicating that Rad51 is a functional and key enzyme of an intrachromosomal recombination pathway. Cells overexpressing Rad51 were resistant to ionizing radiation when irradiated in late S/G2phase of the cell cycle. This suggests that Rad51 participate in the repair of double-strand breaks most likely by homologous recombination involving sister chromatids formed after the S phase.

SUBMITTER: Vispe S

PROVIDER: S-EPMC147643 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation.

Vispé S S Cazaux C C Lesca C C Defais M M

Nucleic acids research 19980601 12

Rad51 proteins share both structural and functional homologies with the bacterial recombinase RecA. The human Rad51 (HsRad51) is able to catalyse strand exchange between homologous DNA molecules in vitro . However the biological functions of Rad51 in mammals are largely unknown. In order to address this question, we have cloned hamster Rad51 cDNA and overexpressed the corresponding protein in CHO cells. We found that 2-3-fold overexpression of the protein stimulated the homologous recombination ...[more]

PMID: 9611228

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Project description:Classical non-homologous end joining (C-NHEJ) and homologous recombination (HR) compete to repair mammalian chromosomal double strand breaks (DSBs). However, C-NHEJ has no impact on HR induced by DNA nicking enzymes. In this case, the replication fork is thought to convert the DNA nick into a one-ended DSB, which lacks a readily available partner for C-NHEJ. Whether C-NHEJ competes with HR at a non-enzymatic mammalian replication fork barrier (RFB) remains unknown. We previously showed that conservative "short tract" gene conversion (STGC) induced by a chromosomal Tus/Ter RFB is a product of bidirectional replication fork stalling. This finding raises the possibility that Tus/Ter-induced STGC proceeds via a two-ended DSB intermediate. If so, Tus/Ter-induced STGC might be subject to competition by C-NHEJ. However, in contrast to the DSB response, where genetic ablation of C-NHEJ stimulates HR, we report here that Tus/Ter-induced HR is unaffected by deletion of either of two C-NHEJ genes, Xrcc4 or Ku70. These results show that Tus/Ter-induced HR does not entail the formation of a two-ended DSB to which C-NHEJ has competitive access. We found no evidence that the alternative end-joining factor, DNA polymerase ?, competes with Tus/Ter-induced HR. We used chromatin-immunoprecipitation to compare Rad51 recruitment to a Tus/Ter RFB and to a neighboring site-specific DSB. Rad51 accumulation at Tus/Ter was more intense and more sustained than at a DSB. In contrast to the DSB response, Rad51 accumulation at Tus/Ter was restricted to within a few hundred base pairs of the RFB. Taken together, these findings suggest that the major DNA structures that bind Rad51 at a Tus/Ter RFB are not conventional DSBs. We propose that Rad51 acts as an "early responder" at stalled forks, binding single stranded daughter strand gaps on the arrested lagging strand, and that Rad51-mediated fork remodeling generates HR intermediates that are incapable of Ku binding and therefore invisible to the C-NHEJ machinery.

Dataset Information

Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation.

Publications

Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation.

Similar Datasets

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