Thursday, April 18, 2024
4/18/2024
Abstract Excess reactive oxygen and nitrogen species (RONS) from radiation, polluted air, as well as chemicals in food and water induce oxidative stress and cause direct damage to nucleotide bases. A prominent form of oxidized damage in the genome and free nucleotide pools is 8-oxoG or 8-oxo-dGTP, respectively. 8-oxoG can hydrogen bond with adenine, resulting in mutations upon DNA replication. 8-oxoG also produces double strand breaks (DSBs) that undergo error-prone repair through non-homologous end-joining (NHEJ). This results in mutagenesis and genomic instability. DNA polymerases mediate repair of oxidative DNA damage. A major obstacle to understanding the etiology and progression of diseases caused by oxidative stress is that the DNA polymerase mechanism employed in the repair of oxidative lesions and effects of accessory factors in the DNA repair complex remain poorly understood. Solving this problem will enable a more complete understanding of the role of DNA polymerases in cancer, aging and disease. The knowledge gained will enable development of therapies to target cancer, neurodegenerative disorders and aging. I will uncover the impact of oxidative stress-induced DNA repair on genome integrity by determining how active site contacts and dynamics in polymerases influence repair outcomes. I will also relate my observations to accessory repair factors and larger repair complexes. To accomplish this task, I will use pH jump crystallography to determine snapshots that will reveal the active site contacts and dynamics employed by NHEJ polymerases ¿ and µ to insert and extend from an 8-oxoG lesion (Aim 1, K99 phase). The functional significance of these contacts will be verified using kinetic assays and mutant enzymes. I hypothesize that these contacts regulate the unique behavior of these polymerases in promoting productive and accurate synthesis and provide insight into the role of 8-oxoG in mutagenesis. I will then employ a combination of transient kinetics and time- lapse crystallography to determine the atomic level contacts and dynamics employed by polymerases ¿ and µ to perform translesion synthesis past and proofreading of the 8-oxoG lesion (Aim 2, R00 phase). The structural intermediates determined in this aim will allow understanding of the structural requirements for mutation prone bypass of 8-oxoG. Building on the results of the latter two aims and training during the K99 period, I will determine the role of substrate channeling among accessory factors during oxidative DNA damage induced NHEJ (Aim 3, R00 phase). Cryo-EM studies will enable determination of how the dynamic and heterogenous populations of NHEJ repair complexes impact oxidative DNA damage repair. I will also determine the structural basis for 8- oxoG processing by Artemis during oxidative NHEJ repair. Completion of this aim will require training in Cryo- EM that will be provided by Dr. Mario Borgnia. The knowledge gained as a result of the work on how polymerases impact repair of oxidative damage and how this mediates productive repair will provide a significant advance in the understanding of how environmental agent induced DNA damage repair. I will gain expertise in state-of-the-art methods such as crystallography, transient kinetics, cryo-EM, DNA replication and DNA repair that will help me achieve my career goals and establish an independent laboratory.
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