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

双链断裂(DSB)是最危险的DNA损伤之一，据估计，在原代人类或小鼠成纤维细胞中，每个细胞每天发生10次。这些自然产生的DSB是在停滞不前的DNA复制叉子崩溃、跨NICK复制、内源性的活性氧物种或DNA内切酶的过早作用时产生的。双链断裂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，用于整个细胞周期，而HR在S/G2期活跃，姐妹染色单体可用作修复模板。DSB修复途径选择的调控对我们的细胞非常重要，因为DSB修复途径选择的错误会导致基因重排，而错误的DSB修复往往会产生异常的染色体结构，从而杀死细胞。NHEJ只在处理最少的DNA末端有活性；它被DNA末端切除抑制，DNA末端切除是5‘-3’核溶解降解产生单链DNA(SsDNA)的过程。复制蛋白A(RPA)蛋白复合体(由~70、32和14 kDa的亚基组成，分别称为RPA70、RPA32和RPA14)立即包被3‘-ssDNA悬垂部分以起保护作用。RPA包被的单链DNA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DNA内切酶。CtIP是多种翻译后修饰(PTM)的靶点，包括磷酸化、泛素化、乙酰化和其他不太为人所知的修饰。蛋白质SUMO化最近成为DSB修复的关键翻译后修饰。一组与HR有关的蛋白质，包括乳腺癌1(BRCA1)、DNA损伤检查点1的介体1(MDC1)、RPA70和Bloom综合征蛋白(BLM)，直接被相扑修饰。尽管蛋白质SUMO化在DNA修复中起着重要作用，但SUMO化是否直接参与DNA末端切除尚不清楚。在本研究中，我们旨在确定SUMO化/去SUMO化如何调节CtIP在DNA末端切除中的功能。在这项应用中，我们发现CtIP在特定的赖氨酸残基上被SUMO化，以响应DNA损伤。在第一个目标中，我们将描述这个苏莫化位点在HR中的功能(S)。在第二个目标中，我们将确定CtIP SUMO化与其他现有的CtIP PTM之间可能的相互作用。我们还鉴定了调节CtIP SUMO化的主要去甲基化酶。在第三个目标中，我们将确定CtIP去SUMO化对其在HR中功能的影响。如果成功，我们的研究将极大地促进我们对DNA末端切除的机制、CtIP SUMO化在DNA修复中的作用以及SUMO化与CtIP的其他PTM之间的相互作用的理解。

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.