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

小泛素样修饰剂 (SUMO) 在 DNA 末端切除中的作用

• 批准号: RGPIN-2017-05752
• 负责人: Ismail, Ismail
• 金额: $ 1.89万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711787
• 关键词: role; small; ubiquitin; like; modifier

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.

双链断裂（DSB）是最危险的DNA损伤之一，据估计在原代人或小鼠成纤维细胞中以每天每个细胞10个的速率发生。这些天然存在的DSB是在停滞的DNA复制叉、跨切口复制、内源性来源的活性氧物质或DNA内切酶的不合时宜的作用崩溃时产生的。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，并在整个细胞周期中使用，而HR在S/G2期活跃，其中姐妹染色单体可用作修复模板。DSB修复途径选择的调节对我们的细胞非常重要，因为DSB修复途径选择中的错误可以产生基因重排，并且错误的DSB修复通常产生杀死细胞的异常染色体结构。NHEJ仅在最低程度加工的DNA末端上有活性;它被DNA末端切除抑制，DNA末端切除是5 '-3'核酸降解产生单链DNA（ssDNA）的过程。3 '-ssDNA突出端立即被复制蛋白A（RPA）蛋白复合物（由约70、32和14 kDa的亚基组成，分别称为RPA 70、RPA 32和RPA 14）包被以进行保护。RPA包被的ssDNA是所有HR子途径的必需中间体。CtBP相互作用蛋白（CtIP）是控制DNA末端切除的关键DNA内切酶。CtIP通过多种翻译后修饰（PTM）靶向，包括磷酸化、泛素化、乙酰化和其他鲜为人知的修饰。 蛋白质SUMO化最近已成为DSB修复的关键PTM。一组参与HR的蛋白质，包括乳腺癌1（BRCA 1），DNA损伤检查点1（MDC 1），RPA 70和布卢姆综合征蛋白（BLM），直接被SUMO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末端切除仍不清楚。在本研究中，我们的目的是确定SUMO化/去SUMO化如何调节CtIP在DNA末端切除中的功能。 在本申请中，我们发现CtIP在响应DNA损伤的特定赖氨酸残基处SUMO化。在第一个目标中，我们将描述HR中SUMO化位点的功能。在第二个目标中，我们将确定CtIP SUMO化和其他现有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SUMO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，我们的研究将大大推进我们对DNA末端切除机制，CtIP SUMO化在DNA修复中的作用以及SUMO化与其他现有CtIP PTM之间的相互作用的理解。