Zhao Bailin, Zhang Dapeng, Li Chengmin, Yuan Zheng, Yu Fangzhi, Zhong Shangwei, Jiang Guibin, Yang Yun-Gui, Le X Chris, Weinfeld Michael, Zhu Ping, Wang Hailin
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences , Beijing, China.
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing, China.
Cell Discov. 2017 Jan 17;3:16053. doi: 10.1038/celldisc.2016.53. eCollection 2017.
Homologous recombination (HR), catalyzed in an evolutionarily conserved manner by active RecA/Rad51 nucleofilaments, maintains genomic integrity and promotes biological evolution and diversity. The structures of RecA/Rad51 nucleofilaments provide information critical for the entire HR process. By exploiting a unique capillary electrophoresis-laser-induced fluorescence polarization assay, we have discovered an active form of RecA nucleofilament, stimulated by ATP hydrolysis, that contains mainly unbound nucleotide sites. This finding was confirmed by a nuclease protection assay and electron microscopy (EM) imaging. We further found that these RecA-unsaturated filaments promote strand exchange and HR . RecA mutants (P67D and P67E), which only form RecA-unsaturated nucleofilaments, were able to mediate HR and , but mutants favoring the formation of the saturated nucleofilaments failed to support HR. We thus present a new model for RecA-mediated HR in which RecA utilizes its intrinsic DNA binding-dependent ATPase activity to remodel the nucleofilaments to a less saturated form and thereby promote HR.
同源重组（HR）由活性RecA/Rad51核丝以进化保守的方式催化，维持基因组完整性，并促进生物进化和多样性。RecA/Rad51核丝的结构为整个HR过程提供了关键信息。通过利用独特的毛细管电泳-激光诱导荧光偏振测定法，我们发现了一种由ATP水解刺激的活性形式的RecA核丝，其主要包含未结合的核苷酸位点。这一发现通过核酸酶保护测定和电子显微镜（EM）成像得到了证实。我们进一步发现，这些RecA不饱和丝促进链交换和HR。仅形成RecA不饱和核丝的RecA突变体（P67D和P67E）能够介导HR，但倾向于形成饱和核丝的突变体则无法支持HR。因此，我们提出了一种RecA介导的HR新模型，其中RecA利用其固有的DNA结合依赖性ATP酶活性将核丝重塑为饱和度较低的形式，从而促进HR。
