REPLICATION RESTART BY RECF RECOMBINATIVE REPAIR

染色体DNA的损伤可以迅速修复，以最大限度地减少复制体对DNA损伤的遭遇，从而导致双链DNA断裂或复制叉子停滞。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分叉崩溃。这些机制涉及重组、修复和复制系统之间的相互作用。例如，修复双链断裂需要RecA、广泛的DNA合成、核酸酶和解旋酶作用来修复断裂和重建复制分叉。该建议侧重于在损坏的站点上复制分叉停顿时发生的事件。RECF重组途径的蛋白质(RecA、F、O、R、J、Q)需要重新启动复制。解决了以下问题：目标I：复制体遇到前导链上的病变后会发生什么？解旋酶是否会继续解开DNA，在病变之前产生单链DNA，以便RecA结合？滞后的单链是继续存在，还是为RecA组装保留了单链DNA？目标二：RecA如何组装到失速的叉子上？在失速的叉子上，RecA作用会产生什么样的链配对中间产物？RECF、O、R、J和Q在这一过程中扮演什么角色？目标III：如何重新启动复制？是复制体吗？ 从搁置的叉子上取下来？它是如何重新组装的？重组是通过原始体还是RECF途径的蛋白质帮助这一过程？如何处理链配对中间产物以重新启动复制？是否需要UvrABC修复机械，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很少。在这项提案中，将详细研究大肠杆菌系统中这些途径的几个蛋白质之间的协调。拟议的研究可能会导致发现DNA新陈代谢的新机制。许多原核生物的DNA代谢机制已被证明适用于真核生物。因此，似乎有理由期待这个项目产生的发现也将成为真核生物中发生的事件的忠实指南。

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.