Department of Molecular Biology & Biotechnology, Krebs Institute, The University of Sheffield, Sheffield, UK.
DNA Repair (Amst). 2011 Feb 7;10(2):138-48. doi: 10.1016/j.dnarep.2010.11.008. Epub 2010 Dec 13.
During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination.
在减数分裂过程中，DNA 双链断裂（DSBs）由同源重组诱导和修复，从而产生基因转换和交叉产物。这些 DSB 主要由 Spo11 产生，它与 DSB 末端共价结合。在酿酒酵母中，很少有其他的减数分裂 DSB 是由自我同源内切酶形成的，如 VDE，它是位点特异性的，并不与 DSB 末端共价结合。我们利用实验定位的 VDE-DSB 位点来分析同源重组的一个中间步骤，即在 DSB 位点处切除单链末端 5'。对具有不同突变等位基因的 MRE11（mre11-58S 和 mre11-H125N）和缺失 EXO1 的菌株进行分析表明，这两种核酸酶对 VDE-DSBs 的修复有重要贡献。对单链修复中间体的物理分析表明，VDE-DSB 处的有效起始和连续性切除需要 Mre11 和 Exo1，任一蛋白质的功能丧失都会导致切除严重延迟。我们提出，这些实验模拟了 Spo11-DSB 去除共价结合蛋白后的情况，并且 Mre11 和 Exo1 是参与产生广泛长度的切除片段的主要核酸酶，这些片段是减数分裂重组的典型特征。
