PROJECT SUMMARY DNA helicases represent one of very few enzyme classes that function in virtually all aspects of DNA replication, recombination, repair, and telomere maintenance. As such, they are vital to maintaining genome integrity and are disease linked when mutated. Thus, there is a critical need to comprehensively understand helicase biology and how these enzymes support genome integrity. 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, both because they are evolutionarily conserved in all domains of life (giving us our pick of model systems to dissect the various aspects of their biology) and because the human PIF1 and RecQ helicases are oncogenes. Indeed, mutations in the genes encoding these helicases are associated with multiple diseases, as well as predispositions to cancers and premature aging. 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. The proposed work will: 1) determine how RecQ4 subfamily helicases and Pso2 family nucleases function in Fanconi anemia- independent DNA ICL repair, identify other factors involved in this repair pathway, and examine how disease alleles of RECQL4 perturb its genome maintenance functions at a mechanistic level; 2) define how PIF1 and RecQ helicases synergistically modulate telomerase activity, determine the impacts of other telomere binding proteins on the biochemistry of these helicases, and reconstitute the telomerase holoenzyme in vitro; and 3) determine the regulatory effects of lysine acetylation on PIF1 family helicases in yeast and humans and how this is linked to genome integrity. 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.