Lee Shauna A, Roques Céline, Magwood Alissa C, Masson Jean-Yves, Baker Mark D
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1 Canada.
DNA Repair (Amst). 2009 Feb 1;8(2):170-81. doi: 10.1016/j.dnarep.2008.10.002. Epub 2008 Nov 18.
The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.
BRCA2肿瘤抑制因子在维持基因组稳定性方面至关重要。有人提出BRCA2可控制核心同源重组蛋白RAD51的可用性、细胞定位及DNA结合活性，BRCA2缺失会导致同源重组缺陷。然而，BRCA2在调节RAD51中的作用以及其他参与RAD51调节的蛋白质（如RAD52和RAD54）相对于BRCA2的功能尚不清楚。在本研究中，我们测试了在Brca2缺失的小鼠杂交瘤细胞中，小鼠Rad51或与Rad51相互作用的小鼠蛋白Rad52和Rad54的表达是否能纠正有缺陷的同源重组。在Brca2缺失的细胞中，野生型小鼠Rad51的过表达可恢复有缺陷的同源重组，但小鼠Rad52或Rad54则不能。同源重组缺陷的纠正需要Rad51的ATP酶活性。相当一部分（约50%）过表达的野生型Rad51定位于细胞核。野生型Rad51过表达在低水平（即无功能）的Brca2存在时恢复同源重组是出乎意料的。我们认为Rad51可能以不依赖Brca2的方式进入细胞核，当Rad51过表达时，在同源重组中对Brca2控制Rad51功能的正常需求是可有可无的。我们的研究支持Rad51功能丧失是Brca2缺失细胞中同源重组缺陷的关键潜在因素。
