REPLICATION RESTART BY RECF RECOMBINATIVE REPAIR

染色体DNA的损伤被迅速修复，以尽量减少复制体遇到的DNA损伤，导致双链DNA断裂，或停止复制叉。然而，这种遭遇不可避免地会发生，细胞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，双链断裂的修复需要RecA、广泛的DNA合成、核酸酶和解旋酶的作用来修复断裂并重建复制叉。此建议侧重于在损坏站点的复制分叉停止时发生的事件。RecF重组途径的蛋白（RecA， F， O， R， J， Q）需要重新开始复制。目的一：当一个复制体在前导链上遇到病变后会发生什么？解旋酶是否继续解开DNA，在病变前产生ssDNA供RecA结合？后链是否继续，或者ssDNA是否保留在那里等待RecA组装？目标二：如何将RecA组装到一个停滞的叉子上？RecA在停叉处的作用会产生哪些链配对中间体？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？是复制体吗

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.