XRCC3 deficiency results in a defect in recombination and increased endoreduplication in human cells

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Abstract
XRCC3 was inactivated in human cells by gene targeting. Consistent with its role in homologous recombination, XRCC3 −/− cells showed a two‐fold sensitivity to DNA cross‐linking agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation and elevated chromosome aberrations. Furthermore, endoreduplication was increased five‐ seven‐fold in the mutants. The T241M variant of XRCC3 has been associated with an increased cancer risk. Expression of the wild‐type cDNA restored this phenotype, while expression of the variant restored the defective recombinational repair, but not the increased endoreduplication. RPA, a protein essential for homologous recombination and DNA replication, is associated with XRCC3 and Rad52. Overexpression of RPA promoted endoreduplication, which was partially complemented by overexpression of the wild‐type XRCC3 protein, but not by overexpression of the variant protein. Overexpression of Rad52 prevented endoreduplication in RPA‐overexpressing cells, in XRCC3 −/− cells and in the variant‐expressing cells, suggesting that deregulated RPA was responsible for the increased endoreduplication. These observations offer the first genetic evidence for the association between homologous recombination and replication initiation having a role in cancer susceptibility.