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.

项目摘要 近紫外线辐射（254 nm光）诱导DNA损伤，阻断复制可能导致 在基因组重排中，当它从错误的位置重新开始时， 不正确的基础是纳入对面的病变，或细胞死亡时，块复制 无法克服在环境诱变剂存在下的不准确复制， 紫外线是负责大多数诱变和重排观察到的癌细胞。 因此，了解如何恢复中断的复制叉对于开发至关重要 用于预防与这些事件相关的不稳定性的治疗剂和策略。在人类中， BRCA 2和RECQ蛋白在维持和处理复制叉中的作用， DNA损伤是众所周知的。然而，复制的机制是 恢复情况尚不清楚。不同的模型表明，无论是修复，translesion 合成或重组可以操作以允许复制恢复。然而，这些途径 并非所有人都能分享同样有益的结果。而DNA修复是无错误的，translesion 合成和重组与突变率和基因组 分别重组。因此，对推进该领域至关重要的是明确确定 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一般恢复机制进行处理 修复酶进入阻塞性损伤并实现修复。大多数体外研究表明 复制体被前导链模板中的DNA损伤破坏，但不被滞后的DNA损伤破坏。 链模板。质粒底物上的进一步前导链损伤优先加工 通过核苷酸切除修复因此，该提案的第一个目标将利用CPD-seq， 建立了高通量测序方法，以绘制紫外线损伤随时间的修复， E.大肠杆菌基因组，并证明前导链损伤优先修复， 复制的该提案的第二个目的是描述holC的作用， 复制体的亚基，在复制偶联修复中发挥作用。holC突变是上位性的， recF突变体，其在被破坏后特别需要处理和恢复复制， 紫外线引起的损伤。此外，据报道HolC（X）与UvrA物理相互作用。因此 这一目标旨在证明，这种相互作用的发生是为了响应 紫外线损伤后，并证明复制偶联修复取决于 HolC的存在。