The role of small ubiquitin-like modifier (SUMO) in DNA end resection

双链断裂（DSBs）是最危险的DNA损伤之一，估计在人类或小鼠原代成纤维细胞中每天每个细胞发生10次。这些自然发生的dsb是在停滞的DNA复制分叉崩溃、切口复制、内源性活性氧或DNA内切酶的不合时宜的作用下产生的。dsb主要通过非同源末端连接（NHEJ）和同源重组（HR）修复。NHEJ是主要途径，在整个细胞周期中使用，而HR在S/G2期活跃，姐妹染色单体可作为修复模板。DSB修复途径选择的调控对我们的细胞非常重要，因为DSB修复途径选择错误会导致基因重排，而DSB修复错误往往会产生异常的染色体结构，从而杀死细胞。NHEJ仅在加工程度最低的DNA末端有活性；DNA末端切除是5‘-3’核溶解降解产生单链DNA （ssDNA）的过程。3'-ssDNA悬悬物立即被复制蛋白A （RPA）蛋白复合物（由~70、32和14 kDa的亚基组成，分别称为RPA70、RPA32和RPA14）包裹以起到保护作用。rpa包被的ssDNA是所有HR亚通路的重要中间体。ctbp相互作用蛋白（CtIP）是控制DNA末端切除的关键核酸内切酶。CtIP是多种翻译后修饰（PTMs）的靶标，包括磷酸化、泛素化、乙酰化和其他鲜为人知的修饰。***蛋白SUMOylation最近被发现是DSB修复的关键PTM。包括乳腺癌1 （BRCA1）、DNA损伤检查点1 （MDC1）、RPA70和Bloom综合征蛋白（BLM）在内的一组与HR相关的蛋白被SUMO直接修饰。虽然很明显，蛋白质SUMOylation在DNA修复中起重要作用，但SUMOylation是否直接参与DNA末端切除尚不清楚。在本研究中，我们的目的是确定SUMOylation/去SUMOylation如何调节CtIP在DNA末端切除中的功能。***在这个应用中，我们发现CtIP在特定赖氨酸残基上被SUMOylated以响应DNA损伤。在第一个目标中，我们将描述HR中这个酰化位点的功能。在第二个目标中，我们将确定CtIP summoylation与其他现有CtIP PTMs之间可能的相互作用。我们还鉴定了调节CtIP summoylation的主要去summoylation酶。在第三个目标中，我们将确定CtIP去sumo化对其在HR中的功能的影响。如果成功，我们的研究将极大地促进我们对DNA末端切除机制的理解，CtIP sumo化在DNA修复中的作用，以及sumo化与CtIP其他现有PTMs之间的相互作用。

Double-strand breaks (DSBs), which are among the most dangerous DNA lesions, are estimated to occur at a rate of ten per cell per day in primary human or mouse fibroblasts. These naturally occurring DSBs are generated upon collapse of stalled DNA replication forks, replication across nicks, reactive oxygen species of endogenous origin or the untimely action of DNA endonucleases. DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary pathway and is used throughout the cell cycle, while HR is active in S/G2 phases where sister chromatids are available as repair templates. The regulation of DSB repair pathway choice is of great importance to our cells since errors in the choice of DSB repair pathway can create gene rearrangements and faulty DSB repair often generates aberrant chromosomal structures that kill cells. NHEJ is active only on minimally processed DNA ends; it is inhibited by DNA end resection, the process by which 5'-3' nucleolytic degradation generates single-stranded DNA (ssDNA). The 3'-ssDNA overhangs are immediately coated by the Replication Protein A (RPA) protein complexes (composed of subunits of ~70, 32, and 14 kDa, referred as to RPA70, RPA32, and RPA14, respectively) for protection. The RPA-coated ssDNA is an essential intermediate of all HR sub-pathways. CtBP-interacting protein (CtIP) is a key DNA endonuclease controlling DNA end resection. CtIP is targeted by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquityation, acetylation and other less known modifications.***Protein SUMOylation has recently emerged as a PTM critical for DSB repair. A group of proteins involved in HR, including Breast Cancer 1 (BRCA1), Mediator Of DNA Damage Checkpoint 1 (MDC1), RPA70, and Bloom syndrome protein (BLM), is directly modified by SUMO. Although it is evident that protein SUMOylation plays an important role in DNA repair, whether SUMOylation is directly involved in DNA end resection is still unclear. In the present study, we aim to determine how SUMOylation/de- SUMOylation regulates CtIP's function in DNA end resection.***In this application, we have found that CtIP is SUMOylated at specific lysine residue in response to DNA damage. In the first objective, we will characterize the function(s) of this sumoylation site in HR. In the second objective, we will determine the possible interplay between CtIP SUMOylation and other existing CtIP PTMs. We also identified the main de-sumoylating enzyme that regulates CtIP SUMOylation. In the third objective, we will determine the impact of CtIP de-SUMOylation on its function in HR. If successful, our studies will greatly advance our understanding of the mechanism of DNA end resection, the role of CtIP SUMOylation in DNA repair, and the interplay between SUMOylation and other existing PTMs of CtIP.