Tomimatsu Nozomi, Mukherjee Bipasha, Harris Janelle Louise, Boffo Francesca Ludovica, Hardebeck Molly Catherine, Potts Patrick Ryan, Khanna Kum Kum, Burma Sandeep
From the Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390.
Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia.
J Biol Chem. 2017 Jun 30;292(26):10779-10790. doi: 10.1074/jbc.M116.772475. Epub 2017 May 17.
End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.
DNA双链断裂（DSB）的末端切除以生成3'-单链DNA，有助于通过无差错的同源重组（HR）进行DSB修复，同时阻碍易错的非同源末端连接（NHEJ）途径的修复。DNA末端切除的激活涉及磷酸肌醇3-激酶样激酶ATM（共济失调毛细血管扩张症突变）和ATR（ATM和Rad3相关）以及细胞周期蛋白依赖性激酶1和2对5'至3'核酸外切酶EXO1的磷酸化。激活后，EXO1也必须受到抑制，以防止过度切除，已知过度切除会妨碍最佳的HR并引发全球基因组不稳定。然而，EXO1受到抑制的机制仍不清楚。在这里，我们报告在人类细胞中DSB诱导后不久，EXO1被泛素-蛋白酶体系统迅速降解。ATR抑制减弱了DNA损伤诱导的EXO1降解，表明ATR介导的EXO1磷酸化将其靶向降解。与这些结果一致，当EXO1中ATR介导的磷酸化所需的SQ基序发生突变时，它对降解产生抗性。我们表明，在DNA损伤诱导后，EXO1被泛素连接酶Skp1-Cullin1-F-box（SCF）家族的成员以磷酸化依赖性方式泛素化。重要的是，抗降解EXO1的表达导致过度切除，这减弱了NHEJ和HR，并严重损害了DSB修复，导致染色体不稳定。这些发现表明，EXO1激活与其最终降解的耦合是一种定时机制，可限制DNA末端切除的程度以进行准确的DNA修复。
