DNA Repair Strategies that Impact Genomic Stability During Oxidative Stress

氧化应激期间影响基因组稳定性的 DNA 修复策略

• 批准号: 9131846
• 负责人: Bret D Freudenthal
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9131846
• 关键词: 8-hydroxyguanosine; 8-oxo-dGTP; Address; Adenine; Air; Base Excision Repairs; Base Pairing; Biological; Breathing; Bypass; Code; Complement; Complex; Coupling; Crystallography; Cytosine; DNA; DNA Damage; DNA Ligases; DNA Polymerase beta; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA-Directed DNA Polymerase; Deoxyguanosine; Drug resistance; Eating; Environmental Exposure; Enzymatic Biochemistry; Epidemiologist; Equilibrium; Excision; Exposure to; Food; Foundations; Generations; Genome Stability; Goals; Health; Human; Kinetics; Lesion; Link; Malignant Neoplasms; Mediating; Mentors; Mentorship; Methods; Modification; Molecular; National Institute of Environmental Health Sciences; Nucleotides; Outcome; Oxidative Stress; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phase; Polymerase; Process; Proteins; Reaction; Repair Complex; Research; Resistance; Risk; Role; Scientist; Solid; Techniques; Testing; Time; Training; United States National Institutes of Health; Vertebral column; base; drinking water; environmental agent; human disease; insight; malignant neurologic neoplasms; mutant; nervous system disorder; new technology; next generation; novel; novel strategies; oxidative DNA damage; phosphodiester; prevent; protein complex; public health relevance; repair enzyme; repaired; response; seal; small molecule; stressor; structural biology; time use

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.

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性应激源引起的。接触会导致DNA损伤，这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-氧代-7，8-二氢-2 '-脱氧鸟苷，其发生在DNA（8-oxoG）和核苷酸池（8-oxo-dGTP）中。8-oxoG和8-oxo-dGTP造成的风险来自其双重编码潜力，导致在DNA聚合酶复制期间与胞嘧啶的非致突变碱基配对或与腺嘌呤的致突变碱基配对。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，允许原子水平的理解聚合酶如何利用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。候选人假设DNA聚合酶（pol）β对氧化性DNA损伤的处理改变了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基本培训，同时确定分子策略，通过该策略，pol Beta使用其逆反应（焦磷酸解）校正相对的8- oxoG。该反应对于基因组稳定性和耐药性具有生物学重要性。结合酶学与时间推移晶体学将确定关键中间体在校对的胞嘧啶或腺嘌呤相对8-oxoG。这将提供分子见解，以调节与8-oxoG相对的诱变腺嘌呤的去除，以增强基因组稳定性或阻断化学治疗链终止药物的去除。在R 00阶段，候选人将确定DNA聚合酶依赖性产生和8-oxoG增殖的分子机制。使用类似的方法，他将确定8-oxo-dGTP是如何插入DNA的，以及8-oxoG在复制过程中是如何被绕过的。这将确定用于处理氧化DNA损伤的分子策略，这些损伤可调节8-oxoG产生和繁殖过程中的致突变结果。候选人将通过确定pol Beta策略对DNA修复过程中辅助因子和途径分化的影响，进一步将自己与导师区分开来。候选人将确定pol β构象变化如何改变底物与其他修复酶的通道（例如，Ape 1）和Ape 1对3 '-8-oxoG的后续加工。 候选人对DNA损伤过程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，他将获得瞬态动力学的基本培训，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损伤是如何处理的，它对更大的修复复合物的影响，并提供对有害人类健康影响的见解，实现了NIEHS-NIH的战略目标。