Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair
Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair
复制标题
DOI：
10.1093/nar/gkv644
复制
发表时间：
2015-09-03
影响因子：
14.9
通讯作者：
Smith, Gerald R.
中科院分区：
文献类型：
--
作者：
Ma, Lijuan;Milman, Neta;Smith, Gerald R.
减数分裂程序性 DNA 双链断裂 (DSB) 修复对于交换和活配子形成至关重要，需要从 5' 末端去除 Spo11-寡核苷酸复合物（剪切）并切除它们以生成侵入性 3' 末端单链 DNA（切除）。两个步骤都需要 Ctp1（Com1、Sae2、CtIP 同源物）与 Mre11-Rad50-Nbs1 (MRN) 复合物共同作用。我们分离了多个粟酒裂殖酵母 ctp1 突变体，这些突变体在减数分裂过程中剪切缺陷但精通切除。值得注意的是，所有突变都聚集在 C 末端部分的保守 CxxC 或 RHR 基序中或附近。通过遗传和物理检测，测试的突变体（如 ctp1 Delta）均存在剪切缺陷。但是，与 ctp1 Delta 不同，这些突变体在 Rec12（Spo11 同源物）独立的断裂修复方面具有重组能力，并且通过物理测定具有切除能力。我们得出结论，细胞内 Ctp1 C 端部分对于剪切至关重要，而 N 端部分足以进行 DSB 末端切除。这一结论与纯化的人 CtIP 切除和核酸内切酶活性是独立的一致。我们的突变体为 Ctp1 的可分离功能提供了细胞内证据。一些突变在与 Seckel 和 Jawad 严重发育综合征相关的 CtIP 突变之一的同一区域截短了 Ctp1，这表明这些综合征是由于需要修复的 DSB 末端缺乏剪切引起的。
Meiotic programmed DNA double-strand break (DSB) repair is essential for crossing-over and viable gamete formation and requires removal of Spo11-oligonucleotide complexes from 5' ends (clipping) and their resection to generate invasive 3'-end single-stranded DNA (resection). Ctp1 (Com1, Sae2, CtIP homolog) acting with the Mre11-Rad50-Nbs1 (MRN) complex is required in both steps. We isolated multiple S. pombe ctp1 mutants deficient in clipping but proficient in resection during meiosis. Remarkably, all of the mutations clustered in or near the conserved CxxC or RHR motif in the C-terminal portion. The mutants tested, like ctp1 Delta, were clipping-deficient by both genetic and physical assays. But, unlike ctp1 Delta, these mutants were recombination-proficient for Rec12 (Spo11 homolog)-independent break-repair and resection-proficient by physical assay. We conclude that the intracellular Ctp1 C-terminal portion is essential for clipping, while the N-terminal portion is sufficient for DSB end-resection. This conclusion agrees with purified human CtIP resection and endonuclease activities being independent. Our mutants provide intracellular evidence for separable functions of Ctp1. Some mutations truncate Ctp1 in the same region as one of the CtIP mutations linked to the Seckel and Jawad severe developmental syndromes, suggesting that these syndromes are caused by a lack of clipping at DSB ends that require repair.