Replication-Coupled Repair: a mechanism for surviving UV irradiation

复制耦合修复：一种在紫外线照射下存活的机制

• 批准号: 10575759
• 负责人: Justin Courcelle
• 金额: $ 18.42万
• 依托单位: PORTLAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10575759
• 关键词: BRCA2 gene; Bacteria; Cell Death; Cell Survival; Cell physiology; Cells; Clinical; Coupled; Coupling; DNA Damage; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; DNA replication fork; Escherichia coli; Event; Genetic Recombination; Genome; Genome Stability; Growth; Health; High-Throughput Nucleotide Sequencing; Homologous Gene; Human; In Vitro; Lesion; Light; Malignant Neoplasms; Maps; Mediating; Microsatellite Instability; Modeling; Mutagenesis; Mutate; Mutation; Nucleotide Excision Repair; Outcome; Pathway interactions; Plasmids; Play; Poly(ADP-ribose) Polymerase Inhibitor; Process; Proteins; Recovery; Replication-Associated Process; Reporting; Role; Therapeutic; Time; UV induced; UV induced DNA damage; Ultraviolet Rays; Work; base; cancer cell; efficacy validation; environmental mutagens; gene product; genome integrity; genome-wide; inhibitor; mutant; prevent; recruit; repair enzyme; repaired; response; targeted cancer therapy; ultraviolet damage; ultraviolet irradiation; ultraviolet lesions

Project Summary Near UV radiation (254nm light) induces DNA damage that blocks replication can result in genomic rearrangements when it resumes from the wrong place, mutagenesis when the incorrect base is incorporated opposite to the lesion, or cell death when the block to replication cannot be overcome. Inaccurate replication in the presence of environmental mutagens such as UV is responsible for the majority of mutagenesis and rearrangements observed in cancer cells. Thus, understanding how disrupted replication forks are restored is critical to developing therapeutics and strategies for preventing instabilities associated with these events. In humans, the role of BRCA2 and RECQ proteins in maintaining and processing replication forks that encounter DNA damage is well known. However, the mechanism by which replication is restored remains unclear. Different models have suggested that either repair, translesion synthesis, or recombination may operate to allow replication to resume. Yet, these pathways do not all share equally beneficial outcomes. Whereas DNA repair is error free, translesion synthesis and recombination are associated with elevated rates of mutations and genome rearrangements respectively. Thus critical to advancing the field, is a clear determination of the mechanism by which DNA replication resumes following disruption. This work will demonstrate that replication forks disrupted by UV-induced DNA damage are primarily processed through a general recovery mechanism that allows nucleotide excision repair enzymes to access to the blocking lesion and effect repair. Most in vitro studies suggest that the replisome is disrupted by DNA lesions in the leading strand template, but not lagging strand template. Further leading strand lesion on plasmid substrates are preferentially processed through nucleotide excision repair. Thus the first aim of this proposal will utilize CPD-seq, an established high-throughput sequencing approach, to map the repair of UV lesions over time on E. coli genome and demonstrate that leading strand lesions are preferentially repaired during replication. The second aim of the proposal characterizes the role that holC, encoding the χ subunit of the replisome, play in the replication-coupled repair. holC mutations are epistatic with recF mutants which is specifically required to process and resume replication after disruption by UV-induced damage. Further, HolC (χ) is reported to physically interact with UvrA. Therefore this aim seeks to and demonstrate that the interaction occurs in response to and functions following UV damage and demonstrate that the replication-coupled repair depends on the presence of holC.

项目概要 近紫外线辐射（254nm 光）会诱导 DNA 损伤，从而阻碍复制 在基因组重排中，当它从错误的位置恢复时，突变发生时 错误的碱基被掺入到病变的对面，或者当复制受阻时细胞死亡 在存在环境诱变剂的情况下无法克服复制不准确的问题。 癌细胞中观察到的大部分突变和重​​排都是由紫外线造成的。 因此，了解如何恢复中断的复制叉对于开发至关重要 预防与这些事件相关的人类不稳定的治疗方法和策略。 BRCA2 和 RECQ 蛋白在维持和处理复制叉中的作用 然而，DNA 损伤的机制是众所周知的。 不同的模型提示修复、损伤是否恢复仍不清楚。 合成或重组可能会使复制恢复，但这些途径确实如此。 并非所有人都能获得同样有益的结果。 合成和重组与突变率和基因组的升高有关 因此，明确确定该领域的发展至关重要。 DNA 复制在中断后恢复的机制。 这项工作将证明复制叉被紫外线诱导的 DNA 损伤破坏 主要通过允许核苷酸切除的通用恢复机制进行处理 大多数体外研究表明，修复酶能够进入阻塞病变并有效修复。 复制体被前导链模板中的 DNA 损伤破坏，但滞后链不会被破坏 优先处理质粒底物上的进一步前导链损伤。 因此，该提案的第一个目标将利用 CPD-seq，一种 建立了高通量测序方法，以绘制紫外线损伤随时间的修复情况 大肠杆菌基因组并证明前导链损伤在 该提案的第二个目标是描述 holC（编码 χ）的作用。 复制体的亚基在复制偶联修复中发挥作用，holC 突变具有上位性。 recF 突变体，特别需要在破坏后处理和恢复复制 此外，据报道 HolC (χ) 与 UvrA 发生物理相互作用。 这一目标旨在并证明互动的发生是为了响应和发挥作用 紫外线损伤后，并证明复制耦合修复取决于 holC 的存在。