Wang Yu, Lamarche Brandon J, Tsai Ming-Daw
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Biochemistry. 2007 May 1;46(17):4962-76. doi: 10.1021/bi0621516. Epub 2007 Apr 4.
In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.
除了将切口/片段化的DNA分子连接回连续的双链外，DNA连接酶还能够通过其对含有错配碱基对的切口的耐受性/不耐受性来影响DNA修复途径的准确性。尽管人类DNA连接酶I（冈崎片段加工）和人类DNA连接酶III/XRCC1复合物（一般DNA修复）已被证明对含有错配碱基对的切口相对不耐受，但人类DNA连接酶IV/XRCC4复合物在这方面尚未得到研究。连接酶IV/XRCC4是唯一通过非同源末端连接（NHEJ）途径参与双链断裂（DSB）修复的DNA连接酶。在化学/物理损伤产生的DSB修复以及V（D）J重组的程序性DSB中间体修复过程中，至少在概念上，存在一些情况，即连接含有错配碱基对的切口的能力似乎是有利的。在此，我们研究连接酶IV/XRCC4是否能为NHEJ贡献错配切口连接活性。为此，我们（i）描述了一种基于大肠杆菌的共表达系统，该系统能提供相对高产率的连接酶IV/XRCC4复合物，（ii）描述了一个独特的限速步骤，这与复合物的检测方式有关，（iii）具体分析XRCC4如何影响连接酶IV的催化作用和底物特异性，以及（iv）通过定量体外动力学分析探究DNA连接酶IV/XRCC4对错配的耐受性/不耐受性。与大多数其他DNA连接酶类似，连接酶IV/XRCC4被证明对含有错配碱基对的切口相当不耐受。根据NHEJ的生物学作用对这些结果进行了讨论。
