Lee Cheng-Sheng, Wang Ruoxi W, Chang Hsiao-Han, Capurso Daniel, Segal Mark R, Haber James E
Department of Biology, Brandeis University, Waltham, MA 02454-9110; Rosenstiel Center, Brandeis University, Waltham, MA 02454-9110;
Center for Communicable Disease Dynamics, Department of Epidemiology, Harvard T. H. Chan School of Public Health, Boston, MA 02115;
Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):E146-54. doi: 10.1073/pnas.1523660113. Epub 2015 Dec 29.
Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5' to 3' resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor.
通过基因转换修复染色体双链断裂（DSB）取决于断裂末端与供体序列相遇的能力。为了了解靶序列的染色体位置如何影响DSB修复，我们利用酵母染色体的全基因组Hi-C分析创建了一系列菌株，其中芽殖酵母中诱导的位点特异性DSB由插入不同位置的2 kb供体序列修复。通过细胞活力或每个供体与参考位点之间的竞争来衡量的修复效率，与每个供体与DSB修复位点的接触频率显示出很强的相关性（r = 0.85和0.79）。修复效率取决于供体和受体之间的距离，而不是特定供体位点的任何内在限制。这些结果进一步证明，寻找同源性是DSB修复中的限速步骤，并表明细胞经常无法修复DSB，因为它们在其他明显致命的过程出现之前无法找到供体。供体位点的修复效率可以通过四个因素提高：DSB末端5'至3'切除速度较慢、复制蛋白因子A（RPA）丰度增加、更长的共享同源性或供体附近存在重组增强元件。
