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 在紫外线辐射附近（254nm的光）诱导DNA损伤，可能会导致复制 在基因组重排时，当它从错误的位置恢复时，诱变 不正确的碱与病变相对或重复块相对或细胞死亡 无法克服。在存在环境诱变剂（例如 紫外线负责癌细胞中观察到的大多数诱变和重排。 那，了解如何恢复复制叉的干扰对于开发至关重要 预防与这些事件相关的不稳定性的治疗和策略。在人类中 BRCA2和RECQ蛋白在维护和加工复制叉中的作用 遇到DNA损伤是众所周知的。但是，复制的机制是 恢复仍然不清楚。不同的模型表明，要么修复，要么 合成或重组可以运行以允许复制恢复。但是，这些途径确实 并非所有人都共享同样有益的结果。而DNA修复是无错误的，translesion 合成和重组与突变率升高和基因组有关 分别重新排列。对领域的前进至关重要，是明确的确定 DNA复制在中断后恢复的机制。 这项工作将表明，紫外线诱导的DNA损伤破坏了复制叉 主要是通过一般恢复机制​​来处理的，该机制使核丁基惊喜 修复酶以进入阻塞病变并修复效果。大多数体外研究表明 重复体在领先的链模板中被DNA病变破坏，但不滞后 链模板。质粒底物上的进一步的前导链病变优选处理 通过核丁基惊喜维修。该提案的第一个目的将利用CPD-Seq，一个 建立的高通量测序方法，以绘制随着时间的推移的修复 大肠杆菌基因组，并证明铅链病变更有可能在 复制。该提案的第二个目的是表征Holc的作用，编码χ 复制体的亚基，在复制耦合维修中播放。 HOLC突变是同志的 RECF突变体，该突变体是特别需要在破坏后处理和恢复复制的必需 紫外线引起的损害。此外，据报道HOLC（χ）与UVRA物理相互作用。所以 此目的旨在并证明相互作用是针对和功能发生的 紫外线损坏后，证明复制耦合维修取决于 存在HOLC。