Mechanism of homology search expansion during recombinational DNA repair

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization.同源搜索（Homology search）是通过同源重组完成DNA双链断裂（double-strand break, DSB）修复的核心步骤。其在细胞内的具体运作机制至今仍有待阐明。本研究开发了一种基于Hi-C的实验方法，可在酿酒酵母（S. cerevisiae）中全基因组范围内绘制单链DNA（single-stranded DNA, ssDNA）的接触图谱，借此揭示了两类主要的同源搜索阶段：由短链Rad51-ssDNA核蛋白纤维（nucleoprotein filaments, NPFs）介导的初始搜索，会通过黏连蛋白（cohesin）介导的染色质环折叠被限制在顺式（cis）作用范围内；刚性核蛋白纤维的渐进性生长使其能够探索远端基因组位点；长距离切除通过提升大规模核蛋白纤维的组装概率，推动了同源搜索从局部到全基因组范围的转变；DNA双链断裂末端锚定可促进反向核蛋白纤维协同开展同源搜索。最终，三号染色体上的自主遗传元件会与核蛋白纤维结合，进而刺激其邻近区域的同源搜索。本研究揭示了由染色质调控因子、长距离切除、末端锚定与特化遗传元件协同调控的同源搜索渐进性扩展机制，同时阐明了其利用Rad51寡聚化所赋予的刚性核蛋白纤维结构的原理。