Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe
Nonhomologous End-Joining with Minimal Sequence Loss Is Promoted by the Mre11-Rad50-Nbs1-Ctp1 Complex in Schizosaccharomyces pombe
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DOI：
10.1534/genetics.117.200972
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发表时间：
2017-05-01
期刊：
影响因子：
3.3
通讯作者：
Runge, Kurt W.
中科院分区：
文献类型：
--
作者：
Li, Yanhui;Wang, Jinyu;Runge, Kurt W.
虽然 Mre11-Rad50-Nbs1 (MRN) 复合物在同源重组和微同源介导的末端连接等修复过程中具有已知的作用，但其在非同源末端连接 (NHEJ) 中的作用尚不清楚，因为酿酒酵母、粟酒裂殖酵母和哺乳动物对修复切割 DNA 末端有不同的要求。大多数双链断裂 (DSB) 需要在 DNA 连接之前进行溶核处理。因此，我们研究了使用 Hermes 转座子的修复，该转座子的切除留下了一个由发夹末端覆盖的 DSB，类似于回文和三核苷酸重复生成的结构。我们使用新型粟酒裂殖酵母瞬时转染系统生成单个 Hermes 插入，并使用 Hermes 切除来显示不可连接末端的 NHEJ 中对 MRN 的需求。 NHEJ 修复表现为缺乏 Ku 或 DNA 连接酶 4 的细胞中切除减少了 1000 倍以上。大多数修复后的切除位点在缺乏每个 MRN 亚基的细胞中减少了 1000 倍，而 MRN 相关 Ctp1 的丢失导致了 30 倍的减少。 Mre11 二聚体被认为将 DNA 末端固定在一起进行修复，而 Mre11 二聚化结构域突变使修复减少了 300 倍。相比之下，内切核酸酶活性缺陷的 mre11 突变体、缺乏 Ctp1 的同一突变体或也缺乏假定的发夹核酸酶 Pso2 的三重突变体显示出野生型修复水平。因此，MRN 可能会招募发夹打开活性，从而进行后续修复。
While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had 1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.