DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress

描述(由申请人提供) 氧化应激是由于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的外源应激源而引起的。暴露会导致DNA损伤，这与癌症和神经疾病的发病机制有关。损伤的主要形式是8-氧-7，8-二氢-2‘-脱氧鸟苷，它同时存在于DNA(8-oxoG)和核苷酸池(8-oxo-dGTP)中。8-oxoG和8-oxo-dGTP的风险源于它们的双重编码潜力，在DNA聚合酶复制过程中，它们导致非突变碱基与胞嘧啶配对或突变碱基与腺嘌呤配对。虽然DNA聚合酶在氧化应激过程中负责调节对人类健康的影响，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，我开发了延时结晶学，使人们能够在原子水平上理解聚合酶如何利用8-oxoG。该方法使用天然底物在反应过程中捕获新的中间体。候选假设是DNA聚合酶(Pola)β对DNA氧化损伤的处理会改变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，候选人将获得瞬变动力学方面的基本培训，同时识别PolBeta利用其反向反应(热磷分解)与8-oxoG相反的分子策略。这一反应对基因组的稳定性和耐药性具有重要的生物学意义。结合酶学和延时结晶学将确定与8-oxoG相反的胞嘧啶或腺嘌呤的校对过程中的关键中间体。这将为调控8-oxoG相对突变腺嘌呤的移除以增强基因组稳定性或阻止化疗链终止药物的移除提供分子洞察力。在R00阶段，候选人将确定依赖DNA聚合酶产生和繁殖8-oxoG的分子机制。使用类似的方法，他将确定如何将8-oxo-dGTP插入DNA，以及如何在复制过程中绕过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的产生和繁殖过程中调节突变结果。候选人将通过确定polBeta策略在DNA修复过程中对辅助因素和途径分化的影响，进一步将自己与他的导师区分开来。候选人将确定polBeta构象变化如何改变底物向其他修复酶(例如APE1)的通道以及APE1对3‘-8-oxoG的后续处理。候选人对DNA损伤处理及其对辅助因素的影响的全面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，他还将接受瞬变动力学方面的基本培训，以补充我的结构生物学背景。这些研究实现了NIEHS-NIH的战略目标，培训了下一代环境科学家，确定了氧化DNA损伤是如何处理的，它对更大的修复共同复合体的影响，并提供了对人类健康有害影响的见解。

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.