PROJECT SUMMARY DNA helicases function in virtually all aspects of DNA replication, recombination, and repair. As such, they are vital to maintaining genome integrity and are disease linked when mutated. Despite many in vivo and in vitro advances in working with helicases, there is a gap in knowledge connecting mutant alleles of helicase genes to the treatment of patients in clinics. The objective of my research is to gain mechanistic insight into how DNA helicases function in genome maintenance and why their dysfunction leads to disease. Toward this goal, we are studying PIF1 and RecQ family helicases, which are evolutionarily conserved and because mutations in the human genes encoding these helicases are associated with multiple diseases. Our current work focuses on the roles of RecQ helicases in DNA inter-strand crosslink (ICL) repair and RecQ and Pif1 helicases in telomere maintenance. To perform this work, we will employ a variety of classic and cutting edge experimental techniques, from standard in vitro enzymatic assays and model organism genetics to next- generation sequencing, crosslinking mass spectrometry, and the development of custom click chemistry probes. Overall, this work will provide fundamental data critical to understanding how PIF1 and RecQ family helicases aid in the maintenance of genome stability, and it will ultimately lead to therapeutic targets and treatments for helicase-linked diseases. I am requesting funds to purchase a four-pack of Cerillo Stratus plate readers with adapter clamps for shaker installation. These plate readers will enable the continuous real-time optical density monitoring of up to 384 independent cultures in four 96-well plates. The universal clamps allow the installation of the Stratus devices in any commercial platform shaker. We routinely perform comparative growth curve analyses of Saccharomyces cerevisiae strains to gauge the effects of mutations, small molecule treatments, and/or heterologous protein expression on the cells. These assays are currently performed in a Biotek Synergy plate reader, but they take between 24 and 48 h of constant use to complete. Therefore, the plate reader is unavailable for other purposes (e.g., DNA and protein quantification), and members of my research group have to sign up weeks in advance to reserve time. The purchase of the Stratus four pack will provide dedicated plate readers for growth curve analyses, improving our throughput and supporting all of our projects.