Oxidative stress is induced by environmental exposure to exogenous stressors found in the air we breathe, food we eat, and water we drink. Exposure leads to DNA damage that is linked to pathogenesis of cancer and neurological disorders. The major form of damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine which occurs in both the DNA (8-oxoG) and nucleotide pools (8-oxo-dGTP). The risk posed by 8-oxoG and 8-oxo-dGTP arises from their dual coding potential resulting in non-mutagenic base pairing with cytosine or mutagenic base pairing with adenine during DNA polymerase replication. While DNA polymerases are responsible for mediating the human health impact during oxidative stress, the strategy they use to process oxidative DNA damage remains unclear. To probe these strategies I have developed time-lapse crystallography, permitting an atomic level understanding of how polymerases utilize 8-oxoG. This approach uses natural substrates to capture novel intermediates during the reaction. The candidate hypothesize that processing of oxidative DNA damage by DNA polymerase (pol) Beta alters DNA repair capacity, impacting downstream accessory factors and repair pathway choice. During the K99 phase, under the mentorship of Dr. Samuel Wilson, the candidate will gain essential training in transient-state kinetics while identifying molecular strategies by which pol Beta proofreads opposite 8- oxoG using its reverse reaction (pyrophosphorolysis). This reaction is biologically important to genomic stability and drug resistance. Combining enzymology with time-lapse crystallography will define key intermediates during the proofreading of cytosine or adenine opposite 8-oxoG. This will provide molecular insights to modulate the removal of the mutagenic adenine opposite 8-oxoG to enhance genomic stability or block the removal of chemotherapeutic chain terminating drugs. In the R00 phase, the candidate will determine the molecular mechanisms of DNA polymerase dependent generation and propagation of 8-oxoG. Using a similar approach, he will determine how 8-oxo-dGTP is inserted into DNA and how 8-oxoG is bypassed during replication. This will identify molecular strategies used to process oxidative DNA damage that modulate the mutagenic outcomes during generation and propagation of 8-oxoG. The candidate will further differentiate himself from his mentor by identifying the impact pol Beta strategies have on accessory factors and pathway differentiation during DNA repair. The candidate will determine how pol Beta conformational changes alter substrate channeling to other repair enzymes (e.g., Ape1) and the subsequent processing of 3'-8-oxoG by Ape1. The candidate's comprehensive study on DNA damage processing and the impact on accessory factors will provide a significant advance to our current understanding of the environmental DNA damage response. Additionally, he will gain essential training in transient-state kinetics to complement my structural biology background. These studies fulfill the strategic goals of the NIEHS-NIH by training the next generation of environmental scientists, determining how oxidative DNA damage is processed, the impact it has on larger repair co-complexes, and providing insights into deleterious human health impacts.
Oxidative stress results from many environmental agents and promotes deleterious modifications to our DNA, ultimately leading to neurological diseases and cancer. This negative cellular impact is mediated by DNA polymerases. To understand the origin of these harmful effects, this proposal uses novel approaches to determine how DNA polymerases process DNA damage, and the influence this has on larger repair complexes. Understanding how oxidative DNA damage arising from environmental exposure is processed will identify novel steps that can be exploited to modulate repair and intervene to enhance human health.
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| Whitaker, Amy M; Schaich, Matthew A; Smith, Mallory R et al. (2017) Base excision repair of oxidative DNA damage: from mechanism to disease. Front Biosci (Landmark Ed) 22:1493-1522 |
| Fouquerel, Elise; Lormand, Justin; Bose, Arindam et al. (2016) Oxidative guanine base damage regulates human telomerase activity. Nat Struct Mol Biol 23:1092-1100 |
