Mohindra Atul, Bolderson Emma, Stone Jason, Wells Michael, Helleday Thomas, Meuth Mark
Institute for Cancer Studies, University of Sheffield, School of Medicine, Sheffield, UK.
Hum Mol Genet. 2004 Jan 15;13(2):203-12. doi: 10.1093/hmg/ddh022. Epub 2003 Nov 25.
Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.
DNA双链断裂（DSB）的修复以及DNA复制叉完整性的维持均需要同源重组修复（HRR）。为了确定在一株HRR缺陷型肿瘤细胞系中发现的RAD51旁系同源物XRCC2的突变等位基因（342delT）的作用，将342delT导入含有重组报告底物的HRR功能正常的细胞中。在一组转染细胞中，342delT的表达使细胞对胸苷和丝裂霉素C敏感，并抑制了由胸苷而非DSB在重组报告基因处诱导的HRR。在另一组转染细胞中，342delT的表达伴随着全长XRCC2水平的降低。这些细胞在由胸苷或DSB诱导的HRR方面存在缺陷。因此，342delT以显性负性方式抑制由胸苷诱导的重组，而由DSB诱导的重组似乎取决于XRCC2的水平以及突变的XRCC2等位基因的表达。这些结果表明，对停滞的复制叉或DSB作出反应的HRR途径在遗传上是可区分的。它们进一步表明XRCC2在复制叉处的HRR中起关键作用，可能是在将RAD51加载到有缺口的DNA上。
