Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining.
Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining.
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DOI：
10.1093/nar/gkw013
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发表时间：
2016-04-07
影响因子：
14.9
通讯作者：
Wilson TE
中科院分区：
文献类型：
--
作者：
Liang Z;Sunder S;Nallasivam S;Wilson TE
非同源末端连接（Non-homologous end joining， NHEJ）是有限5 '切除细胞DNA双链断裂（DSBs）的主要修复途径。为了更好地了解染色体DSB的悬垂极性如何影响NHEJ，我们使用优化的锌指核酸酶在酵母中制备了位点特异性的5 ‘悬垂DSB (5 ’ DSB)，其效率接近ho诱导的3 ' DSB的形成。当控制DSB形成的程度时，修复监测表明染色体5 ' DSB比3 ' DSB更有效地重新连接，这与NHEJ蛋白在5 ' DSB上的强大募集一致。连接介导的qPCR显示，Mre11- rad50 - xrs2可以快速修饰5 ' dsb并促进对3 ' dsb的保护，这可能是通过Mre11核酸酶识别悬空极性来实现的。新一代测序结果显示，5 ‘ dsb上的NHEJ突变频率更高，验证了3 ’和5 ' dsb上Pol4聚合酶的差异需求。末端加工酶Tdp1不像以前的质粒研究那样影响染色体5 ' dsb的连接保真度，尽管Tdp1仅以ku独立的方式被招募到5 ' dsb上。这些结果表明，基于悬垂极性的不同DSB处理方式通过DSB修饰酶的不同招募和作用影响NHEJ动力学和保真度。
Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5′ resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5′-overhanging DSBs (5′ DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3′ DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5′ DSBs were rejoined more efficiently than 3′ DSBs, consistent with a robust recruitment of NHEJ proteins to 5′ DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5′ DSBs and facilitated protection of 3′ DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5′ DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3′ and 5′ DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5′ DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5′ DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.