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.

通过同源重组（HR）这一无错通路完成的DNA双链断裂修复，需要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下游反应必不可少的关键中间产物。本研究证实，EXO1是小泛素样修饰物（SUMO）的靶标，且SUMO修饰会影响EXO1的泛素化水平与蛋白质稳定性。我们鉴定出一条依赖于UBC9-PIAS1/PIAS4的调控机制，可在体内介导人类EXO1的SUMO修饰，并通过体外重构体系证实，酿酒酵母中由Ubc9-Siz1/Siz2介导的该调控机制具有保守性。此外，我们还在体外与体内环境中均检测到EXO1与去SUMO化酶SENP6之间存在物理相互作用，该作用可增强EXO1的稳定性。最后，我们确定了EXO1上主要的SUMO修饰位点，并证实异位表达SUMO修饰缺陷型EXO1，可挽救野生型（wt）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。