Huertas Pablo, Cortés-Ledesma Felipe, Sartori Alessandro A, Aguilera Andrés, Jackson Stephen P
The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
Nature. 2008 Oct 2;455(7213):689-92. doi: 10.1038/nature07215. Epub 2008 Aug 20.
DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.
DNA双链断裂（DSB）通过两种主要机制进行修复：非同源末端连接（NHEJ）和同源重组（HR）。HR是最精确的DSB修复机制，但通常限于细胞周期的S期和G2期，此时DNA已经复制，姐妹染色单体可作为修复模板。相比之下，NHEJ在整个细胞周期中都起作用，但在G1期最为重要（参考文献4、6）。修复途径之间的选择由细胞周期蛋白依赖性蛋白激酶（CDK）控制，主要控制点在DSB切除水平，这一事件是HR所必需的，但不是NHEJ所必需的，并且在S期和G2期最有效地发生（参考文献2、5）。在这里，我们确定酿酒酵母中DSB切除的细胞周期控制是由CDK对Sae2蛋白中一个进化保守基序的磷酸化引起的。我们表明，将Sae2的Ser 267突变为不可磷酸化的残基会导致与sae2Delta缺失突变体相当的表型，包括对喜树碱过敏、孢子形成缺陷、发夹诱导的重组减少、DNA末端加工严重受损以及HR因子的组装和拆卸错误。此外，模拟组成型Ser 267磷酸化的Sae2突变可补充这些表型，并克服CDK活性对DSB切除的必要性。Sae2突变还会导致细胞周期阶段特异性的DNA损伤超敏反应，并影响HR和NHEJ之间的平衡。因此，这些发现为DSB修复的细胞周期控制提供了一个机制基础，并突出了调节DSB切除的重要性。
