DNA helicases and associated factors in genome stability

点击翻译按钮获取中文摘要

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 Refeyn TwoMP mass photometer. This device enables rapid mass measurements of small volumes of dilute biomolecules, with single-molecule precision. We routinely generate recombinant protein preparations for biochemical assays, and knowing the oligomeric state and monodispersity under physiological conditions is critical to the design, analysis, and interpretation of our experiments. We currently use size-exclusion chromatography (SEC) to determine recombinant protein mass, but SEC is slow, has low resolution, and requires large volumes of very concentrated protein. Therefore, SEC limits the availability of our chromatography system and is incompatible with proteins that cannot be purified in large amounts or do not tolerate the concentration process. The purchase of a mass photometer will provide a rapid and high-resolution solution to these problems with a dedicated device that is inexpensive and easy to use, improving our biophysical capabilities and supporting all of our projects.