Ruff Patrick, Donnianni Roberto A, Glancy Eleanor, Oh Julyun, Symington Lorraine S
Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA.
Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA.
Cell Rep. 2016 Dec 20;17(12):3359-3368. doi: 10.1016/j.celrep.2016.12.003.
DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.
DNA双链断裂（DSB）是具有细胞毒性的损伤，必须进行精确修复以维持基因组稳定性。复制蛋白A（RPA）通过保护由末端切除形成的单链DNA（ssDNA）中间体并促进Rad51装载，在DSB的同源依赖性修复中发挥重要作用。我们发现，支持染色体内同源重组的RFA1次等位基因突变体在涉及长片段DNA合成的修复过程中，特别是在断裂诱导复制（BIR）方面存在严重缺陷。通过消除Sgs1-Dna2切除途径，可以部分抑制rfa1突变体的BIR缺陷，这表明当RPA没有完全保护时，Dna2核酸酶会攻击末端切除过程中形成的ssDNA。还发现过表达Rad51可以抑制rfa1的BIR缺陷。我们认为，Rad51与过度末端切除形成的ssDNA以及D环迁移过程中的ssDNA结合，可以部分补偿功能失调的RPA。
