Mechanism and regulation of DNA double-strand break repair

DNA双链断裂修复机制及调控

• 批准号: 10623591
• 负责人: Lorraine S Symington
• 金额: $ 69.54万
• 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 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623591
• 关键词: Address; Biological Models; Cell Survival; Cells; Cellular Metabolic Process; Chemotherapy and/or radiation; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Chromosomal Breaks; Complex; Coupled; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Gene; DNA biosynthesis; DNA replication fork; Defect; Development; Disease; Double Strand Break Repair; Endonuclease I; Event; Excision; Genetic Recombination; Genomic Instability; Goals; Human; Immunologics; Investigation; Knowledge; Lesion; Malignant Neoplasms; Methodology; Neurologic; Normal Cell; Nucleosomes; Pathway interactions; Predisposition; Process; Proteins; Regulation; Research; Resistance; Role; Saccharomyces cerevisiae; Sister Chromatid; Site; System; Toxic effect; Yeasts; chromatin remodeling; chromosome loss; cytotoxic; genome integrity; homologous recombination; insight; novel; novel therapeutics; practical application; programs; repaired; replication stress; targeted treatment

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, 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. Homologous recombination (HR), one of the two main mechanisms to repair DSBs, employs extensive homology and templated DNA synthesis to restore the broken chromosome. In addition to its role in repairing DSBs, HR has emerged as a prominent mechanism to restart stalled or collapsed replication forks. 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 end resection, the first step of HR, while more extensive processing of the 5 terminated strands is catalyzed by Exo1 or Dna2- Sgs1. Specifically, we will use S1-seq and MNase-seq methodologies to determine whether chromatin remodeling precedes or is coupled to end resection, and will determine which resection mechanisms are impacted by loss of RSC, SNF, INO80 and Fun30 chromatin remodelers. The second part of our program focuses on the repair single-end DSBs produced by collapse of replication forks. We recently developed an efficient system to create a site-specific replication fork collapse using Cas9 nickase and have shown that cell survival is completely dependent on the MRX complex and Rad51. This system will allow us to define the elusive functions of MRX in sister-chromatid recombination and to identify novel factors that participate in collapsed fork repair. In the long term we believe that mechanisms under investigation in this proposal will provide new insight into genomic instability caused by replication stress and how chromatin structure modulates DSB repair.

染色体双链断裂(DSB)是在正常细胞过程中自发发生的细胞毒性损伤 用DNA损伤剂对细胞进行新陈代谢或随后的处理。如果未修复或修复不当， DSB可导致严重的有害事件，如染色体丢失、缺失或易位。缺陷 在DSB的修复过程中会引起基因组的不稳定，表现为免疫性、发育性或神经性 缺陷和癌症的易感性。DSB的毒性也被用于放射和化疗。 作为针对特定DNA修复蛋白的靶向治疗。因此，了解这一机制 DSB修复对开发新的治疗方法和治疗方法具有重要意义和实际应用 揭示了抵抗的途径。同源重组(HR)，修复的两种主要机制之一 DSB，利用广泛的同源性和模板DNA合成来修复断裂的染色体。在……里面 除了在修复DSB方面的作用外，HR已经成为重启停滞或崩溃的重要机制 复制分叉。我们研究计划的总体目标是破译同源依赖的机制 DSB修复，以酿酒酵母为模型系统。我们计划的第一部分构建 我们以前的研究表明，保守的Mre11-Rad50-Xrs2(MRX)复合体启动末端切除， HR的第一步，而5个末端链的更广泛的加工是由外显子1或DNA 2催化的- 军士1。具体地说，我们将使用s1-seq和mNase-seq方法来确定染色质 重建先于末端切除或与末端切除相结合，并将决定哪些切除机制是 受RSC、SNF、INO80和Fun30染色质重构体缺失的影响。我们节目的第二部分 重点介绍了复制叉崩溃产生的单端DSB的修复。我们最近开发了一种 使用Cas9 Nickase创建特定部位复制叉状折叠的高效系统，并显示了该细胞 生存完全依赖于MRX复合体和RAD51。这个系统将允许我们定义难以捉摸的 MRX在姐妹染色单体重组中的作用及寻找参与分叉折叠的新因子 修理。从长远来看，我们相信，这项提案中正在调查的机制将提供新的见解 复制应激引起的基因组不稳定性以及染色质结构如何调节DSB修复。

项目成果

Interstitial telomere sequences disrupt break-induced replication and drive formation of ectopic telomeres.

• DOI: 10.1093/nar/gkaa1081
• 发表时间: 2020-12-16
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Stivison EA;Young KJ;Symington LS
• 通讯作者: Symington LS

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

• DOI: 10.7554/elife.84322
• 发表时间: 2023-06-30
• 期刊: eLife
• 影响因子: 7.7
• 作者: Kimble MT;Johnson MJ;Nester MR;Symington LS
• 通讯作者: Symington LS

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper-resection.

磷酸化蛋白质组学揭示了 DNA 末端超切除后独特的 Mec1/ATR 信号反应。

• DOI: 10.15252/embj.2020104566
• 发表时间: 2021
• 期刊: The EMBO journal
• 作者: Sanford,EthanJ;Comstock,WilliamJ;Faça,VitorM;Vega,StephanieC;Gnügge,Robert;Symington,LorraineS;Smolka,MarcusB
• 通讯作者: Smolka,MarcusB

Efficient DNA double-strand break formation at single or multiple defined sites in the Saccharomyces cerevisiae genome.

• DOI: 10.1093/nar/gkaa833
• 发表时间: 2020-11-18
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Gnügge R;Symington LS
• 通讯作者: Symington LS