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，估计在人类或小鼠原代成纤维细胞中每个细胞每天会发生 10 次双链断裂。这些天然存在的 DSB 是在停滞的 DNA 复制叉崩溃、跨切口复制、内源性活性氧或 DNA 核酸内切酶的不合时宜作用时产生的。 DSB主要通过非同源末端连接（NHEJ）和同源重组（HR）来修复。 NHEJ 是主要途径，在整个细胞周期中使用，而 HR 在 S/G2 期活跃，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 修复途径选择错误会导致基因重排，而错误的 DSB 修复通常会产生杀死细胞的异常染色体结构。 NHEJ 仅在经过最少加工的 DNA 末端有活性；它受到 DNA 末端切除的抑制，DNA 末端切除是 5'-3' 溶核降解生成单链 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 RPA70、RPA32 和 RPA14）包被以进行保护。 RPA 包被的 ssDNA 是所有 HR 子通路的重要中间体。 CtBP 相互作用蛋白 (CtIP) 是控制 DNA 末端切除的关键 DNA 核酸内切酶。 CtIP 是多种翻译后修饰 (PTM) 的靶标，包括磷酸化、泛素化、乙酰化和其他鲜为人知的修饰。 蛋白质 SUMO 化最近已成为对 DSB 修复至关重要的 PTM。 SUMO 直接修饰了一组与 HR 相关的蛋白质，包括乳腺癌 1 (BRCA1)、DNA 损伤检查点介导物 1 (MDC1)、RPA70 和布卢姆综合征蛋白 (BLM)。尽管蛋白质 SUMO 化在 DNA 修复中发挥着重要作用，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。 在此应用中，我们发现 CtIP 在特定的赖氨酸残基处发生 SUMO 化，以响应 DNA 损伤。在第一个目标中，我们将描述 HR 中该苏酰化位点的功能。在第二个目标中，我们将确定 CtIP SUMOylation 和其他现有 CtIP PTM 之间可能的相互作用。我们还确定了调节 CtIP SUMOylation 的主要去苏酰化酶。在第三个目标中，我们将确定 CtIP 去 SUMOylation 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、CtIP SUMOylation 在 DNA 修复中的作用以及 SUMOylation 与 CtIP 其他现有 PTM 之间相互作用的理解。