DNA helicases and associated factors in genome stability

项目总结

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 an Odyssey CLx Imaging System with a D-Digit attachment. This imager enables two-channel (700 and 800 nm) near-infrared fluorescence and chemiluminescent imaging of membranes, gels, plates, slides, and even large samples such as organs. The D-Digit further extends functionality to the scanning, imaging, and analysis of agarose DNA gels stained with ethidium bromide or a variety of safe stains (e.g., GelRed) without the need for ultraviolet transillumination. We routinely perform gel- based assays to assess protein expression, protein-protein interactions, protein-nucleic acid interactions, and the biochemical activities of DNA helicases and associated proteins, often using expensive radioisotopes for labeling. Further, multiple pieces of shared equipment spread across buildings on campus are necessary to visualize and analyze our experiments, and our time on the equipment is limited. The purchase of the Odyssey CLx and D-Digit will provide a dedicated, isotope-free solution to our gel-based imaging and analysis needs.