Zhao Lingyun, Xu Jingfei, Zhao Weixing, Sung Patrick, Wang Hong-Wei
Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
Yale University, New Haven, CT, United States.
Methods Enzymol. 2018;600:179-199. doi: 10.1016/bs.mie.2017.12.002. Epub 2018 Feb 1.
Homologous recombination is a universal tool for DNA double-strand break and replication fork repair, and it is catalyzed by a highly conserved family of recombinases. In eukaryotes, Rad51 is the recombinase that catalyzes the pairing of homologous DNA molecules and the exchange of strands between the paired molecules. Rad51 assembles on single-stranded DNA (ssDNA) stemming from lesion processing to form a right-handed helical polymer that engages then samples double-stranded DNA (dsDNA) for homology. Upon matching with a homologous sequence, the Rad51-bound ssDNA invades the dsDNA, leading to the formation of a DNA joint with concomitant displacement of the strand of like polarity. The Rad51-DNA filaments are amenable to structural studies using cryo-electron microscopy (cryo-EM). In particular, recent technical breakthroughs in cryo-EM have made it possible to define the structure and function of human RAD51 at near-atomic resolution. In this chapter, we describe our cryo-EM approach to capture the human RAD51 filament structures in various stages of catalysis. The approach may also be useful for related recombinases and other helical assemblies.
同源重组是一种用于修复DNA双链断裂和复制叉的通用工具，它由一个高度保守的重组酶家族催化。在真核生物中，Rad51是催化同源DNA分子配对以及配对分子间链交换的重组酶。Rad51在损伤处理产生的单链DNA（ssDNA）上组装，形成右手螺旋聚合物，该聚合物随后与双链DNA（dsDNA）进行同源性匹配。与同源序列匹配后，与Rad51结合的ssDNA侵入dsDNA，导致形成DNA接头并伴随相同极性链的置换。Rad51-DNA细丝适合使用冷冻电子显微镜（cryo-EM）进行结构研究。特别是，冷冻电子显微镜最近的技术突破使得以近原子分辨率定义人类RAD51的结构和功能成为可能。在本章中，我们描述了我们用于捕获处于催化不同阶段的人类RAD51细丝结构的冷冻电子显微镜方法。该方法可能对相关重组酶和其他螺旋组装体也有用。
