REPLICATION RESTART BY RECF RECOMBINATIVE REPAIR

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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.