Shivji Mahmud K K, Mukund Shreyas R, Rajendra Eeson, Chen Shaoxia, Short Judith M, Savill Jane, Klenerman David, Venkitaraman Ashok R
The Medical Research Council Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ,United Kingdom.
Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13254-9. doi: 10.1073/pnas.0906208106. Epub 2009 Jul 23.
The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2([BRC1-8])). Here, we show that BRCA2([BRC1-8]) exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2([BRC1-8]) alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2([BRC1-8]) promotes RAD51 loading onto ssDNA. In contrast, BRCA2([BRC1-8]) has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2([BRC1-8]) stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing recombination products. Collectively, our findings suggest that BRCA2([BRC1-8]) targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.
乳腺癌和卵巢癌抑制因子BRCA2在同源DNA重组（HDR）过程中控制酶RAD51，以维持基因组稳定性。BRCA2通过分散在一个1127个残基区域（BRCA2([BRC1-8])）中的8个保守BRC重复基序与RAD51结合。在这里，我们表明BRCA2([BRC1-8])对RAD51与单链（ss）和双链（ds）DNA底物的结合产生相反的影响，增强链交换。BRCA2([BRC1-8])改变了与ssDNA底物结合的RAD51的电泳迁移率，电子显微镜显示，同时伴随着与ssDNA结合的蛋白质组装体的增加。单分子荧光光谱表明，BRCA2([BRC1-8])促进RAD51加载到ssDNA上。相比之下，BRCA2([BRC1-8])对RAD51在dsDNA上的组装有不同的影响；它抑制并减缓了这个过程。当同源ssDNA和dsDNA同时存在时，BRCA2([BRC1-8])刺激链交换，随着代表重组产物的接头分子的出现，RAD51加载到dsDNA上的过程延迟。总的来说，我们的研究结果表明，BRCA2([BRC1-8])将RAD51靶向ssDNA，同时抑制dsDNA结合，并且这些相反的活性相互支持，以刺激HDR。我们的工作为人类HDR机制及其受BRCA2肿瘤抑制因子调控提供了新的见解。
