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Defining the mechanism of Shieldin complex in DNA end joining

定义 Shieldin 复合物在 DNA 末端连接中的机制

基本信息

批准号：
10456078
负责人：
Joshua Heyza
金额：
$ 6.98万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-12 至 2023-08-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456078
关键词：
Address Affinity BRCA1 gene BRCA2 gene Binding Biological Biological Assay CRISPR therapeutics CRISPR/Cas technology Cell Cycle Cell Line Cell physiology Cells Chemicals Clinical Clinical Research Complex DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair Dangerousness Defect Development Disease Double Strand Break Repair Event Excision Foundations Future G2 Phase Genes Genetic Genome Stability Human Immune Immune System Diseases Immunoglobulin Class Switching Immunoglobulin Switch Recombination In Vitro Lasers Lead Length Lesion Malignant Neoplasms Maps Mechanics Mediating Mediator of activation protein Meiosis Modeling Molecular Mutation Nonhomologous DNA End Joining Outcomes Research Pathway interactions Physiological Processes Poly(ADP-ribose) Polymerases Proteins Resistance Resolution Role S phase Signal Transduction Sister Chromatid Syndrome Work cancer cell genome editing genome integrity homologous recombination human disease imaging approach improved in vivo inhibitor insight live cell imaging molecular imaging novel anticancer drug novel therapeutic intervention p53-binding protein 1 preservation protein protein interaction recruit repaired response single molecule tumor

项目摘要

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.

项目概要：由于外源性和内源性化学物质，人类细胞经常获得DNA双链断裂（DSB）以及作为自然生理过程（例如免疫类别转换重组和减数分裂交叉）的一部分事件）。因此，细胞已经进化出高度调节的DSB修复途径，其用于保护免受尤其是对基因组完整性的威胁。正是由于这个原因，对于正确的DSB识别，处理和解决有助于各种人类疾病，包括免疫紊乱、各种遗传综合征和癌症发展。一般来说，细胞可以获得 DSB拆分的两个主要途径，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53 BP 1蛋白，其通过阻断NHEJ来促进NHEJ。在DSB处进行广泛切除，这将有利于HR。Shieldin复合物（由SHLD 1，SHLD 2， SHLD 3和REV 7）最近被鉴定为53 BP 1信号传导的下游效应子。Shieldin效应53 BP 1's 抗HR通过结合DSB处的ssDNA突出端和阻断末端切除而发挥功能。这些观察导致以下难题：NHEJ更喜欢短突出端或钝DNA末端，如何通过 ssDNA结合复合物我建议定义屏蔽蛋白在DSB中功能的分子机制使用单分子（SM）成像方法进行修复。为此，我将生成一组HaloTagged DNA 使用CRISPR/Cas9介导的基因组编辑的损伤反应（DDR）蛋白，将用于SM活细胞，细胞成像有了这些标记的蛋白质，我将剖析控制Shieldin的调节机制，本发明的目的是在体内将Shieldin募集至DNA DSB，并在体外定义Shieldin在DSB处结合的分子决定簇。产生Shieldin功能在调节DSB修复中的定量模型将揭示DSB修复的基本原理。 Shieldin如何控制DSB修复途径选择的机制。因此，这些研究的结果将具有由于Shieldin在DSB修复中的作用尚不完全清楚，因此在该领域可能产生重大影响。此外，生成Shieldin功能的定量模型可能会导致揭示新的靶向Shieldin功能的治疗方法以获得临床益处。