REPLICATION RESTART BY RECF RECOMBINATIVE REPAIR

通过 RECF 重组修复重新启动复制

• 批准号: 6498874
• 负责人: MICHAEL E O'DONNELL
• 金额: $ 37.58万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498874
• 关键词: DNA damage; DNA repair; DNA replication; DNA replication origin; Escherichia coli; bacterial DNA; bacterial proteins; exonuclease; genetic recombination; helicase; nucleic acid metabolism

Damage to chromosomal DNA is quickly repaired to minimize encounter of DNA damage by the replisome, resulting in double strand DNA breaks, or a stalled replication fork. However, inevitably such encounters occur and the cell has additional mechanisms to handle the resulting replication fork collapse. These mechanisms involve interplay among recombination, repair and replication systems. For example, repair of double strand breaks require RecA, extensive DNA synthesis, nuclease and helicase action to mend the break and reestablish replication forks. This proposal focuses on events that transpire upon stalling of the replication fork at a damaged site. Proteins of the RecF recombination pathway (RecA, F, O, R, J, Q) are required to restart replication. The following questions are addressed: Aim I: What happens after a replisome runs into a lesion on the leading strand? Does the helicase continue to unwind DNA, generating ssDNA ahead of the lesion for RecA to bind? Does the lagging strand continue, or is ssDNA preserved there for RecA assembly? Aim II: How does RecA assemble onto a stalled fork? What strand pairing intermediates result from RecA action at a stalled fork? What roles do RecF, O, R, J and Q play in the process? Aim III: How is replication restarted? Is the replisome removed from the stalled fork? How is it reassembled? Is reassembly via the primosome or do proteins of the RecF pathway assist the process? How are strand pairing intermediates processed for restarting replication? Is the UvrABC repair machinery required, and are branch migrating enzymes needed? Very little biochemical information exists on how replication, recombination and repair pathways interrelate. Coordination among the several proteins of these pathways in the E. coli system will be examined in detail in this proposal. The proposed studies will likely lead to discovery of new mechanisms in DNA metabolism. Many prokaryotic DNA metabolic mechanisms have been shown to generalize to eukaryotes. Therefore, it seems reasonable to expect that discoveries emanating from this project will serve as a faithful guide for events that occur in eukaryotes as well.

染色体 DNA 的损伤可以快速修复，以最大程度地减少复制体对 DNA 的损伤，从而导致双链 DNA 断裂或复制叉停滞。然而，这种遭遇不可避免地会发生，并且细胞有额外的机制来处理由此产生的复制叉崩溃。这些机制涉及重组、修复和复制系统之间的相互作用。例如，双链断裂的修复需要 RecA、广泛的 DNA 合成、核酸酶和解旋酶作用来修复断裂并重建复制叉。该提案重点关注复制叉在受损站点停滞时发生的事件。 RecF 重组途径的蛋白质（RecA、F、O、R、J、Q）是重新启动复制所必需的。解决以下问题： 目标 I：复制体遇到前导链上的损伤后会发生什么？解旋酶是否会继续解旋 DNA，在损伤之前生成 ssDNA 以供 RecA 结合？滞后链是否继续，或者 ssDNA 是否保留在那里用于 RecA 组装？目标 II：RecA 如何组装到停滞的前叉上？ RecA 在停滞叉处的作用会产生什么链配对中间体？ RecF、O、R、J、Q在此过程中扮演什么角色？目标三：复制如何重新启动？是复制体吗 从停滞的货叉中取出？它是如何重新组装的？重组是通过引发体还是 RecF 途径的蛋白质协助该过程？如何处理链配对中间体以重新启动复制？是否需要 UvrABC 修复机制，是否需要分支迁移酶？ 关于复制、重组和修复途径如何相互关联的生化信息非常少。本提案将详细研究大肠杆菌系统中这些途径的几种蛋白质之间的协调。拟议的研究可能会导致 DNA 代谢新机制的发现。许多原核 DNA 代谢机制已被证明可以推广到真核生物。因此，我们有理由期望该项目的发现也将成为真核生物中发生的事件的忠实指南。