DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress

描述（由申请人提供） 氧化应激是由环境暴露于我们呼吸的空气、我们吃的食物和我们喝的水中发现的外源压力源引起的。接触会导致 DNA 损伤，而 DNA 损伤与癌症和神经系统疾病的发病机制有关。损伤的主要形式是 8-oxo-7,8-二氢-2'-脱氧鸟苷，它发生在 DNA (8-oxoG) 和核苷酸库 (8-oxo-dGTP) 中。 8-oxoG 和 8-oxo-dGTP 带来的风险源于它们的双重编码潜力，导致在 DNA 聚合酶复制过程中与胞嘧啶发生非诱变碱基配对，或与腺嘌呤发生诱变碱基配对。虽然 DNA 聚合酶负责介导氧化应激期间对人类健康的影响，但它们用于处理氧化 DNA 损伤的策略仍不清楚。为了探索这些策略，我开发了延时晶体学，可以在原子水平上了解聚合酶如何利用 8-oxoG。这种方法使用天然底物在反应过程中捕获新的中间体。候选者假设 DNA 聚合酶 (pol) Beta 对氧化性 DNA 损伤的处理会改变 DNA 修复能力，影响下游辅助因子和修复途径的选择。在 K99 阶段，在 Samuel Wilson 博士的指导下，候选人将获得瞬态动力学方面的必要培训，同时确定 pol Beta 使用其逆反应（焦磷酸解）校对相反 8-oxoG 的分子策略。该反应对于基因组稳定性和耐药性具有重要的生物学意义。将酶学与延时晶体学相结合将在校对 8-oxoG 相对的胞嘧啶或腺嘌呤期间定义关键中间体。这将为调节与 8-oxoG 相对的诱变腺嘌呤的去除提供分子见解，以增强基因组稳定性或阻止化疗链终止药物的去除。在 R00 阶段，候选人将确定 8-oxoG 依赖于 DNA 聚合酶的生成和传播的分子机制。使用类似的方法，他将确定 8-oxo-dGTP 如何插入 DNA 以及复制过程中如何绕过 8-oxoG。这将确定用于处理氧化 DNA 损伤的分子策略，这些策略在 8-oxoG 的生成和繁殖过程中调节诱变结果。候选人将通过确定 pol Beta 策略对 DNA 修复过程中辅助因素和途径分化的影响，进一步将自己与导师区分开来。候选人将确定 pol Beta 构象变化如何改变底物通道至其他修复酶（例如 Ape1）以及 Ape1 对 3'-8-oxoG 的后续处理。 该候选人对 DNA 损伤处理及其对辅助因素影响的综合研究将为我们目前对环境 DNA 损伤反应的理解提供重大进展。此外，他还将获得瞬态动力学方面的必要培训，以补充我的结构生物学背景。这些研究通过培训下一代环境科学家、确定氧化 DNA 损伤的处理方式、其对更大的修复复合物的影响以及提供对人类健康有害影响的见解，实现了 NIEHS-NIH 的战略目标。

DESCRIPTION (provided by applicant) 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.