PROJECT SUMMARY DNA damage cannot be prevented because elements in a normal cellular environment including water and oxygen contribute to damage. Therefore, DNA repair pathways are essential for maintaining genomic stability and preventing diseases associated with DNA damage-induced mutagenesis. Even with functional DNA repair pathways, some DNA damage will inevitably escape repair and block DNA replication when encountered by the replisome. Cells have specialized pathways to bypass or repair DNA damage that blocks replication to allow replication to proceed. DNA helicases are among the enzymes essential for DNA repair, and these enzymes act both during and outside of DNA replication. The Rad3/XPD family of iron-sulfur (Fe-S)-containing DNA helicases plays an important role in DNA repair and maintaining genome stability. There are four family members in humans, XPD, FANCJ (a.k.a. BRIP1), DDX11 (a.k.a. ChlR1), and RTEL1, that are crucially important for human health as evidenced by diseases associated with mutations in each of the genes. Genetic disorders linked to mutations in Rad3/XPD family helicases are typically associated with genome instability, a predisposition to cancer, and a number of other pathologies. This proposal will define biochemical and molecular mechanisms for a newly discovered member of the Rad3/XPD helicase family in Escherichia coli, YoaA, that plays a role in repairing DNA damage during DNA replication. Our collaborators in the Lovett laboratory use 3’-azido- 3’deoxythymidine (AZT) as a reagent to block DNA replication in E. coli, and they identified two genes, yoaA and holC, that work together to give cells tolerance to AZT. Protein sequence predicts, and our preliminary results confirm, that the first gene, yoaA, encodes an Fe-S helicase. The second gene, holC, encodes the c subunit of DNA polymerase III holoenzyme (pol III HE) implicating the E. coli replicase in repair of AZT lesions. However, our preliminary results have uncovered a novel function for c as a subunit of the YoaA helicase, and we propose that c functions with the YoaA helicase rather than pol III HE in a pathway that repairs AZT lesions. The main premise of this proposal is that YoaA and c constitute a DNA helicase that is involved in the repair of damaged 3’ ends at stalled replication forks. Our aims are to: 1) characterize the YoaA•c protein, 2) characterize the helicase and substrate preferences for YoaA•c, and 3) define functional interactions between YoaA and c in vitro and develop a key reagent to investigate these functional interactions in vivo. This proposal will provide the first biochemical characterization of the YoaA•c helicase, a member of the Rad3/XPD family of helicases which play critical roles in human health by maintaining genome stability.