Sumoylation regulates EXO1 stability and processing of DNA damage

DNA double-strand break repair by the error-free pathway of homologous recombination (HR) requires the concerted action of several factors. Among these, EXO1 and DNA2/BLM are responsible for the extensive resection of DNA ends to produce 3′-overhangs, which are essential intermediates for downstream steps of HR. Here we show that EXO1 is a SUMO target and that sumoylation affects EXO1 ubiquitylation and protein stability. We identify an UBC9-PIAS1/PIAS4-dependent mechanism controlling human EXO1 sumoylation in vivo and demonstrate conservation of this mechanism in yeast by the Ubc9-Siz1/Siz2 using an in vitro reconstituted system. Furthermore, we show physical interaction between EXO1 and the de-sumoylating enzyme SENP6 both in vitro and in vivo, promoting EXO1 stability. Finally, we identify the major sites of sumoylation in EXO1 and show that ectopic expression of a sumoylation-deficient form of EXO1 rescues the DNA damage-induced chromosomal aberrations observed upon wt-EXO1 expression. Thus, our study identifies a novel layer of regulation of EXO1, making the pathways that regulate its function an ideal target for therapeutic intervention.

借助同源重组（homologous recombination，HR）的无错通路修复DNA双链断裂，需多种因子协同发挥作用。其中，EXO1与DNA2/BLM负责对DNA末端进行广泛切除，以生成3'单链突出端——这是HR下游步骤不可或缺的中间产物。本研究发现EXO1是SUMO（small ubiquitin-like modifier，小泛素相关修饰物）的靶标蛋白，且SUMO化修饰可影响EXO1的泛素化修饰与蛋白质稳定性。我们鉴定出一条依赖UBC9-PIAS1/PIAS4的调控机制，可在体内对人源EXO1的SUMO化修饰进行调控，并通过体外重构实验体系，证实酵母中Ubc9-Siz1/Siz2介导的该调控机制具有进化保守性。此外，我们还证实EXO1与去SUMO化酶SENP6在体外与体内均存在物理相互作用，该作用可促进EXO1的蛋白质稳定性。最后，我们鉴定出EXO1上的主要SUMO化修饰位点，并证实异位表达SUMO化缺陷型EXO1，能够挽救野生型EXO1表达时所观察到的DNA损伤诱导染色体畸变。综上，本研究揭示了EXO1调控的全新层级，使得调控其功能的通路成为治疗干预的理想靶点。