DNA helicases and associated factors in genome stability

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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. In this application, I am requesting funds to support a 10-week research experience for Ms. Morgan Roush in my lab during the summer of 2023. The purpose of this project is to enable Ms. Roush to gain research experience in molecular biology and yeast genetics to support her goals to pursue a career in biomedical research. Ms. Roush’s research project will examine the link between G-quadruplex (G4) DNA stability and genome integrity. Ms. Roush aims to generate Saccharomyces cerevisiae strains lacking the enzymatic activity of one or more DNA helicases implicated in G4 structure unwinding and maintenance. She will then test the effects of over-expressing a G4-binding and -stabilizing nanobody in these strains to compare the toxicity of G4 stabilization to wild-type cells containing a full complement of helicases. Ms. Roush will attend and present at our weekly group meeting, and she will present a poster in the Indiana University Undergraduate Research Symposium at the end of summer 2023. It is my further hope that her summer research experience is positive and that she will remain a productive member of my lab until she graduates.