Replication fork repair is essential for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. The long-term goal of this study is a more complete mechanistic understanding of the repair of replication gaps and how such processes are regulated. Objectives will be to elucidate elements of replication gap repair pathways, including specific replisome components, using genetic and biochemical experiments. Because all cells repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organism, Escherichia coli, should reveal mechanisms applicable to repair of DNA in human cells. The use of azidothymidine in this study as a compound to induce replication gaps has the potential to reveal toxicity and tolerance mechanisms relevant to its therapeutical use as an anti-HIV agent. In addition, because the DNA damage response in bacterial pathogens plays a role in toxin production, antibiotic resistance and persistence of infection, this work could provide new targets for antibiotic therapy.
The first aim of this study seeks to clarify how the YoaA protein, a member of the XPD/FANCJ family of helicases mutated in human disease syndromes, participates in replication gap repair by studies of its genetic and biochemical properties. The central hypothesis of this study is that interactions with the replisome protein, Chi, recruits YoaA helicase to a stalled replication fork and that its unwinding of the nascent strand facilitates removal of chain-terminating lesions. Effects on specific repair pathways will be tested.
The second aim of this proposal is to identify the mechanism and extent of the SOS-independent DNA damage response. Many replication and repair genes share a common feature of DNA damage inducibility independent of the SOS response.
This aim will center on the iraD gene, discovered as a regulatory factor required for survival to replication inhibition and oxidative damage. The role of the DnaA protein in this regulation and signaling via replication clamps will be examined by in vivo and in vitro experiments. This study will significantly advance the field of DNA repair by providing new information regarding how replication gaps are sensed and repaired.
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