REPLICATION RESTART BY RECF RECOMBINATIVE REPAIR

对染色体DNA的损伤被快速修复以最小化复制体对DNA损伤的遭遇，从而导致双链DNA断裂或停滞的复制叉。然而，不可避免地会发生这样的遭遇，并且单元具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。此建议侧重于在损坏站点停止复制分叉时发生的事件。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。以下问题得到解决：目的一：发生了什么后，复制体运行到病变的领导链？解旋酶是否继续解开DNA，在损伤之前产生ssDNA供RecA结合？滞后链是否继续存在，或者ssDNA是否被保留在那里用于RecA组装？目标二：RecA如何组装到停滞的叉子上？什么链配对中间体的结果从RecA行动在一个停滞的叉？RecF、O、R、J和Q在这个过程中扮演什么角色？目标三：如何重新启动复制？是复制体 从停滞的叉子上取下来它是如何重新组装的？重组是通过原始体还是RecF途径的蛋白质协助这一过程？如何处理链配对中间体以重新开始复制？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物化学信息非常少。在E.大肠杆菌系统的可行性进行详细研究。拟议的研究可能会导致发现DNA代谢的新机制。许多原核生物的DNA代谢机制已被证明可以推广到真核生物。因此，似乎有理由期待，从这个项目产生的发现将作为一个忠实的指导事件发生在真核生物以及。

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.