San Filippo Joseph, Chi Peter, Sehorn Michael G, Etchin Julia, Krejci Lumir, Sung Patrick
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
J Biol Chem. 2006 Apr 28;281(17):11649-57. doi: 10.1074/jbc.M601249200. Epub 2006 Mar 2.
BRCA2 likely exerts its tumor suppressor function by enhancing the efficiency of the homology-directed repair of injured chromosomes. To help define the DNA repair role of BRCA2, we expressed and purified a polypeptide, BRC3/4-DBD, that harbors its BRC3 and BRC4 repeats and DNA binding domain. BRC3/4-DBD interacted with hRad51 and bound DNA with a distinct preference for single-stranded (ss) DNA. Importantly we demonstrated by biochemical means and electron microscopy that BRC3/4-DBD nucleates hRad51 onto ssDNA and acts as a recombination mediator in enabling hRad51 to utilize replication protein A-coated ssDNA as recombination substrate. These functions of BRC3/4-DBD required both the BRC repeats and the BRCA2 DNA binding domain. The results thus clarify the role of BRCA2 in Rad51-dependent DNA recombination and repair, and the experimental strategies described herein should be valuable for systematically deciphering this BRCA2 function.
BRCA2可能通过提高受损染色体同源定向修复的效率来发挥其肿瘤抑制功能。为了帮助确定BRCA2在DNA修复中的作用，我们表达并纯化了一种多肽BRC3/4-DBD，它含有BRC3和BRC4重复序列以及DNA结合结构域。BRC3/4-DBD与hRad51相互作用，并以对单链（ss）DNA有明显偏好的方式结合DNA。重要的是，我们通过生化方法和电子显微镜证明，BRC3/4-DBD能使hRad51在ssDNA上成核，并作为重组介质，使hRad51能够将复制蛋白A包被的ssDNA用作重组底物。BRC3/4-DBD的这些功能既需要BRC重复序列，也需要BRCA2 DNA结合结构域。因此，这些结果阐明了BRCA2在依赖Rad51的DNA重组和修复中的作用，本文所述的实验策略对于系统地解读这种BRCA2功能应该是有价值的。
