Mechanism and regulation of DNA double-strand break repair

DNA双链断裂修复机制及调控

• 批准号: 10407594
• 负责人: Lorraine S Symington
• 金额: $ 61.68万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10407594
• 关键词: Address; Alleles; Biological Assay; Biological Models; Cell Cycle; Cells; Cellular Metabolic Process; Chemotherapy and/or radiation; Chromatin Structure; Chromosomes; Complex; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA damage checkpoint; DNA replication fork; Defect; Development; Disease; Double Strand Break Repair; Event; Excision; Genetic; Genomic Instability; Goals; Human; Immunologics; Investigation; Knowledge; Laboratories; Lead; Length; Lesion; Malignant Neoplasms; Measures; Neurologic; Nonhomologous DNA End Joining; Normal Cell; Pathway interactions; Phosphotransferases; Predisposition; Process; Regulation; Research; Resistance; Saccharomyces cerevisiae; Site; Toxic effect; Yeasts; chromosome loss; cytotoxic; genome integrity; homologous recombination; insight; next generation sequencing; novel therapeutics; practical application; programs; recombinational repair; recruit; repaired; targeted treatment; telomere

Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or following treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to profoundly detrimental events, such as chromosome loss, deletions, duplications or translocations. Defects in the repair of DSBs cause genomic instability, manifested as immunological, development or neurological defects, and predisposition to cancer. The toxicity of DSBs is exploited for radiation and chemotherapy, as well as targeted therapies directed against specific DNA repair proteins. Thus understanding the mechanisms of DSB repair is of fundamental importance and has practical application for development of new therapeutics and uncovering pathways to resistance. Typically, cells repair DSBs by either homologous recombination (HR) or non-homologous end joining (NHEJ). HR employs extensive homology and templated DNA synthesis to restore the broken chromosome and is considered to be an error-free process. NHEJ directly ligates DSB ends, a mechanism that is potentially error prone due to small deletions or insertions at the junctions. The choice between these two pathways is governed by the cell cycle, which regulates an early step in HR, namely, 5'-3' resection of DSBs. The overall goal of our research program is to decipher the mechanisms of homology-dependent DSB repair, using the yeast Saccharomyces cerevisiae as a model system. The first part of our program builds on our previous studies showing that the conserved Mre11- Rad50-Xrs2 (MRX) complex initiates 5'-3' resection. Specifically, we will use next-generation sequencing to identify the sites of MRX nicking, determine how chromatin structure influences nick site selection and measure the length of resection tracts in cells undergoing HR repair. In addition to controlling end resection, MRX promotes NHEJ, tethers DSB ends and recruits the Tel1ATM kinase to DSBs to activate the DNA damage checkpoint. We will determine the contribution of these diverse functions to genome integrity using specific alleles of MRX components coupled to assays measuring gross chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are repaired by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). The second part of our research program utilizes physical and genetic assays developed in my laboratory to address the mechanism and fidelity of DNA synthesis by BIR.

染色体双链断裂（DSB）是在正常细胞周期中自发发生的细胞毒性损伤。 在某些实施方案中，细胞可以通过代谢或在用DNA损伤剂处理细胞之后进行。如果未经修理或修理不当， DSB可导致严重的有害事件，如染色体丢失、缺失、复制或突变。 易位DSB修复缺陷导致基因组不稳定，表现为免疫学， 发育或神经缺陷，以及癌症易感性。DSB的毒性被用于 放疗和化疗，以及针对特定DNA修复蛋白的靶向治疗。因此 了解DSB修复的机制具有根本的重要性，并具有实际应用， 开发新的治疗方法和发现耐药途径。通常，细胞通过以下方式修复DSB： 同源重组（HR）或非同源末端连接（NHEJ）。HR采用广泛的同源性， 模板化DNA合成以恢复断裂的染色体，并且被认为是无错误的过程。 NHEJ直接连接DSB末端，这是一种由于小的缺失或缺失而可能容易出错的机制。 在连接处插入。这两种途径之间的选择是由细胞周期， 调节HR的早期步骤，即DSB的5 '-3'切除。我们研究计划的总体目标是 利用酿酒酵母作为一种新的基因工程工具， 模型系统。我们计划的第一部分建立在我们以前的研究基础上，表明保守的Mre 11- Rad 50-Xrs 2（MRX）复合物启动5 '-3'切除。具体来说，我们将使用下一代测序技术， 确定MRX切口位点，确定染色质结构如何影响切口位点选择， 测量进行HR修复的细胞中切除道的长度。除了控制末端切除， MRX促进NHEJ，束缚DSB末端并将Tel 1ATM激酶募集至DSB以激活DNA损伤 检查站我们将确定这些不同的功能，基因组完整性的贡献，使用特定的 MRX组分的等位基因偶联至测量总染色体重排的测定。出现的争端解决机构 通过复制叉崩溃或通过无帽端粒的侵蚀，只有一个自由端， 链侵入同源双链DNA，随后复制到染色体末端（断裂诱导 复制，BIR）。我们的研究计划的第二部分利用物理和遗传分析， 我的实验室，以解决机制和保真度的DNA合成的BIR。