PROJECT SUMMARY DNA replication is frequently blocked by DNA damage or other obstacles (e.g. transcription complexes) that must be overcome to allow replication to continue. Specialized pathways are essential for rescuing stalled DNA replication forks to allow replication to proceed. Our collaborators in the Lovett laboratory use 3’-azido- 3’deoxythymidine (AZT) as a reagent to block DNA replication in Escherichia coli, and they identified two genes, yoaA and holC, that work together to give cells tolerance to AZT. Protein sequence predicts, and our biochemical results confirm, that the first gene, yoaA, encodes an iron-sulfur (Fe-S)-containing DNA helicase in the Rad3/XPD family. The second gene, holC, encodes the c subunit of the DNA polymerase III holoenzyme and we have uncovered a novel function for c as a subunit of the YoaA helicase. DNA helicases, including the Rad3/XPD family of Fe-S-containing DNA helicases, play essential roles in maintaining genome stability by participating in DNA repair and rescuing stalled replication forks. There are four Rad3/XPD family members in humans, XPD, FANCJ (a.k.a. BRIP1), DDX11 (a.k.a. ChlR1), and RTEL1, that are crucially important for human health. Genetic disorders linked to mutations in these helicases are typically associated with genome instability, a predisposition to cancer, and a number of other pathologies. Our parent proposal defines biochemical and molecular mechanisms for the newly discovered E. coli family member, YoaA. The main premise 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 activities and substrate preferences for YoaA•c, and 3) determine how c contributes to YoaA activities in vitro, and to develop a cy fusion protein for assaying functions of c as a subunit of YoaA versus the DNA polymerase III holoenzyme in vivo. Our parent 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. This equipment supplement requests the purchase of a LUMICKS M-trap optical tweezer system with wide-field fluorescence to measure helicase activities of YoaA•c (and YoaA) at the single-molecule level. This purchase will directly support the experiments in Aims 2 and 3 of our parent proposal investigating the mechanism of YoaA and contributions that c makes to the helicase activities of YoaA. To date, XPD is the only Rad3/XPD family member that has been characterized using this type of single-molecule methodology. Identifying similarities and differences in the activities of these family members is important for understanding their functions under normal conditions and dysfunction in disease states. Moreover, this single-molecule approach is highly complementary to the ensemble experiments that we are...