Defining the mechanism of Shieldin complex in DNA end joining

项目总结： 由于外源性和内源性化学物质，人类细胞定期获得DNA双链断裂(DSB) 作为自然生理过程的一部分(例如，免疫类开关重组和减数分裂交换 事件)。因此，细胞进化出高度调控的DSB修复途径，用于保护 对基因组完整性的威胁尤其危险。正是由于这个原因，通路中的突变导致了 正确的DSB识别、处理和解析有助于多种人类疾病，包括 免疫紊乱、各种遗传综合征和癌症的发展。通常，单元格可以访问 DSB拆分的两条主要途径是同源重组(HR)和非同源末端连接 (NHEJ)。DSB修复途径选择的关键调节因子是53BP1蛋白，它通过阻断NHEJ来促进NHEJ 广泛切除DSB有利于HR。Shieldin络合物(由SHLD1，SHLD2， SHLD3和Rev7)是53BP1信号的下游效应因子。希尔丁效应53BP1‘S 抗HR作用是通过结合DSB上的单链DNA悬垂和阻断末端切除来实现的。这些观察结果导致了 对于以下难题：喜欢短悬挑或钝化DNA末端的NHEJ如何通过 单链DNA结合复合体？我建议定义DSB中Shieldin功能的分子机制 使用单分子(SM)成像方法进行修复。为此，我将生成一个HaloTaged DNA面板 使用CRISPR/Cas9介导的基因组编辑的损伤响应(DDR)蛋白，将用于SM Live- 细胞成像。利用这些标记的蛋白质，我将剖析控制Shieldin的调控机制 在体内向DNADSB募集，并确定体外DSB上Shieldin结合的分子决定因素。 建立Shieldin在调节DSB修复中的作用的定量模型将揭示基本的 Shieldin如何控制DSB修复途径选择的机制。因此，这些研究的结果将会有 由于Shieldin在DSB修复中的作用，对该领域潜在的重大影响尚不完全清楚。 此外，生成Shieldin函数的量化模型可能会导致新的 以Shieldin功能为靶点的治疗方法使临床受益。

Project Summary: Human cells regularly acquire DNA double strand breaks (DSB) due to exogenous and endogenous chemicals and as part of natural physiological processes (e.g. immune class switch recombination and meiotic crossover events). Therefore, cells have evolved highly regulated DSB repair pathways which serve to protect from particularly dangerous threats to genome integrity. It is for this reason that mutations in pathways responsible for proper DSB recognition, processing, and resolution contribute to a variety of human diseases including immune disorders, a variety of genetic syndromes, and cancer development. In general, cells have access to two major pathways for DSB resolution, homologous recombination (HR) and non-homologous end joining (NHEJ). A key regulator of DSB repair pathway choice is the 53BP1 protein which promotes NHEJ by blocking extensive resection at a DSB which would favor HR. The Shieldin complex (composed of SHLD1, SHLD2, SHLD3 & REV7) was recently identified as a downstream effector of 53BP1 signaling. Shieldin effects 53BP1’s anti-HR functions by binding ssDNA overhangs at DSBs and blocking end resection. These observations lead to the following conundrum: how can NHEJ, which prefers short overhangs or blunt DNA ends, be promoted by a ssDNA binding complex? I propose to define the molecular mechanism underlying Shieldin function in DSB repair using single-molecule (SM) imaging approaches. To this end, I will generate a panel of HaloTagged DNA damage response (DDR) proteins using CRISPR/Cas9 mediated genome editing that will be used for SM live- cell imaging. With these tagged proteins, I will dissect the regulatory mechanisms that control Shieldin recruitment to DNA DSBs in vivo and define the molecular determinants for Shieldin binding at DSBs in vitro. Generating a quantitative model of Shieldin function in regulating DSB repair will reveal the fundamental mechanics of how Shieldin controls DSB repair pathway choice. As such, results from these studies will have the potential for significant impact in the field as Shieldin’s role in DSB repair is incompletely understood. Additionally, generating a quantitative model of Shieldin function could lead to the uncovering of novel therapeutic approaches to target Shieldin function for clinical benefit.