Kwon Youngho, Zhao Weixing, Sung Patrick
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA.
Methods Mol Biol. 2011;745:421-35. doi: 10.1007/978-1-61779-129-1_24.
Rad51-mediated pairing between homologous DNA sequences during homologous recombination (HR) plays pivotal roles in DNA double-strand break repair. The multi-step process occurs through cooperation of Rad51 and a number of accessory protein factors. The development of various biochemical analyses with the requisite purified factors provides an opportunity to understand the molecular mechanisms of HR. In this chapter, we describe detailed procedures of in vitro assays using human Rad51, a polypeptide derived from the BRCA2 protein, and the Hop2-Mnd1 complex, to examine (1) homologous DNA pairing, (2) Rad51 targeting to single-stranded DNA, (3) stabilization of the Rad51 nucleoprotein filament, and (4) duplex capture by the Rad51 nucleoprotein filament. These methods are invaluable for delineating the functional interplay of HR factors.
在同源重组（HR）过程中，Rad51介导的同源DNA序列配对在DNA双链断裂修复中起着关键作用。这个多步骤过程通过Rad51与许多辅助蛋白因子的协作来发生。利用所需的纯化因子进行各种生化分析的发展，为理解HR的分子机制提供了契机。在本章中，我们描述了使用人Rad51、一种源自BRCA2蛋白的多肽以及Hop2-Mnd1复合物进行体外测定的详细程序，以检测（1）同源DNA配对，（2）Rad51靶向单链DNA，（3）Rad51核蛋白丝的稳定，以及（4）Rad51核蛋白丝对双链的捕获。这些方法对于描绘HR因子的功能相互作用非常重要。
