Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable.
Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable.
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DOI：
10.1093/nar/gkw557
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发表时间：
2016-09-30
影响因子：
14.9
通讯作者：
Russell P
中科院分区：
文献类型：
--
作者：
Jensen KL;Russell P
同源重组（HR）修复程序性减数分裂双链断裂（DSB）需要从5′ DNA末端内切核酸剪切Rec 12 Spo 11-寡核苷酸，然后切除以产生侵入性3′单链DNA尾。Mre 11-Rad 50-Nbs 1（MRN）内切核酸酶和Ctp 1（CtIP和Sae 2直系同源物）是裂殖酵母中两种活性所必需的，但它们是否在遗传上是可分离的是有争议的。在这里，我们调查有丝分裂DSB修复特性的Ctp 1 C-末端结构域（ctp 1-CD）突变体，据报道是专门剪切缺陷。这些突变体对许多断裂剂敏感，包括那些产生缺乏共价结合蛋白的DSB的断裂剂。这些敏感性被抑制基因消除Ku非同源末端连接（NHEJ）蛋白，表明Ctp 1依赖性剪切MRN是需要Ku从DNA末端去除。然而，这种拯救需要Exo 1切除活性，这意味着MRN的Ctp 1依赖性切除在Ctp 1-CD突变体中是有缺陷的。ctp 1-CD突变体耐受一个但不是多个断裂的复制叉，并且它们高度依赖于Chk 1介导的细胞周期检查点阻滞，表明HR修复是低效的。我们的结论是Ctp 1的C-末端结构域是需要有效的剪辑和切除DSB的MRN和这些活动是机械相似的。
Homologous recombination (HR) repair of programmed meiotic double-strand breaks (DSBs) requires endonucleolytic clipping of Rec12Spo11-oligonucleotides from 5′ DNA ends followed by resection to generate invasive 3′ single-stranded DNA tails. The Mre11-Rad50-Nbs1 (MRN) endonuclease and Ctp1 (CtIP and Sae2 ortholog) are required for both activities in fission yeast but whether they are genetically separable is controversial. Here, we investigate the mitotic DSB repair properties of Ctp1 C-terminal domain (ctp1-CD) mutants that were reported to be specifically clipping deficient. These mutants are sensitive to many clastogens, including those that create DSBs devoid of covalently bound proteins. These sensitivities are suppressed by genetically eliminating Ku nonhomologous end-joining (NHEJ) protein, indicating that Ctp1-dependent clipping by MRN is required for Ku removal from DNA ends. However, this rescue requires Exo1 resection activity, implying that Ctp1-dependent resection by MRN is defective in ctp1-CD mutants. The ctp1-CD mutants tolerate one but not multiple broken replication forks, and they are highly reliant on the Chk1-mediated cell cycle checkpoint arrest, indicating that HR repair is inefficient. We conclude that the C-terminal domain of Ctp1 is required for both efficient clipping and resection of DSBs by MRN and these activities are mechanistically similar.
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通讯作者：
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