Revealing DNA Polymerase Molecular Strategies in End-Joining Double Strand Break Repair - PROJECT SUMMARY/ABSTRACT DNA double strand breaks (DSBs) are highly cytotoxic and threaten the physical integrity of the genome. Repair of the majority of DSBs through nonhomologous end joining (NHEJ) is thus essential to cellular survival and function. Erroneous end joining can lead to mutagenesis and physical chromosomal changes that promote adverse human health outcomes and disease. Oxidative stress, generated by exposure to damaging agents in the cellular environment, is a prevalent source of cellular DSBs and produces chemically diverse structurally modified breaks and ends. These modified breaks modulate enzymatic end processing steps in DSB repair in a poorly understood manner, altering the processing and channeling of repair intermediates, and thus the molecular repair strategies and pathways employed to repair specific breaks. When NHEJ is challenged or blocked, highly erroneous repair pathways, such as microhomology mediated end joining, that often lead to mutagenesis and physical chromosomal changes prevalent in cancers, are recruited to complete repair. DSB repair polymerases and nucleases are crucial in break processing of especially damaged and modified ends and breaks. Despite progress in uncovering key features of accurate and erroneous repair, the molecular strategies of polymerases and nucleases in DSB repair remain poorly understood. This proposed MIRA effort will reveal detailed polymerase and nuclease atomic and molecular end processing mechanisms through a comprehensive approach that integrates cutting-edge structural biology, mechanistic biochemistry, biophysics, computational, and molecular biology. We have three related overarching goals: to uncover the mechanisms of DSB repair polymerases and nucleases in end joining, to determine how these mechanisms modulate the efficiency and fidelity of repair within the repair complex, and to reveal the effects of oxidative modifications of break substrates on repair strategies. The proposed projects will 1) elucidate the molecular fidelity strategies of DSB repair polymerases and nucleases and the impact of oxidative damage at authentic DSBs using time-resolved crystallography, mechanistic biochemistry, molecular and computational biology, 2) reveal the protein complex and the effects of the repair architecture on polymerase and nuclease functions using molecular biology, mechanistic biochemistry and cryo-EM, 3) uncover steps along repair that can possibly be modulated in a therapeutic setting. I am in a unique position to advance these efforts due to my strong track record in DNA repair, scientific experience and expertise, network of collaborators, outstanding institutional resources, and my multidisciplinary approach. Successful completion of these aims will resolve longstanding questions about fundamental molecular DSB repair strategies, especially upon oxidative modification. This work will thus provide transformative insights into DSB repair, and the effects of environmental stress on repair strategies, that will inform approaches to modulate repair in mechanism, biology, and disease.
