Structural and Mechanistic Studies of DNA Repair

DNA修复的结构和机制研究

• 批准号: 9762147
• 负责人: Bret D Freudenthal
• 金额: $ 38.25万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762147
• 关键词: Address; Affect; Base Excision Repairs; Biological; Cell model; Cells; Complex; Crystallography; DNA; DNA Damage; DNA Repair; DNA Structure; DNA polymerase A; DNA-Directed DNA Polymerase; Dangerousness; Development; Drug Combinations; Drug Design; Enzyme Kinetics; Enzymes; Exonuclease; Foundations; Genome Stability; Genomic Instability; Goals; Hand; Health; Human; Individual; Location; Malignant Neoplasms; Methodology; Microscopy; Modification; Molecular; Multiprotein Complexes; Mutagenesis; Neutrons; Oxidative Stress; Pathway interactions; Positioning Attribute; Process; Proteins; Protons; RNA; Reaction; Repair Complex; Ribonucleotides; Role; Scientific Advances and Accomplishments; Structure; Therapeutic; Time; X-Ray Crystallography; base; biophysical techniques; cancer cell; deviant; human disease; interdisciplinary approach; novel therapeutics; oxidative DNA damage; repair enzyme; repaired; response; simulation; single molecule; therapeutic development

Oxidative stress is a prevalent and dangerous cellular condition resulting in deleterious modifications to the structure of DNA. These modifications promote mutagenesis and consequently the development of numerous human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against oxidative DNA damage and is a vital guardian of genome stability. While the roles of individual enzymes during a classical BER cycle are largely established, it remains enigmatic how these enzymes function together in a multi-protein/DNA complex to facilitate the channeling of toxic DNA repair intermediates between each protein. In addition, it is poorly understood how deviations in the classical BER pathway affect the DNA repair process and genome stability. These deviancies range from mismatched-, damaged-, and ribo-nucleotides inserted by a DNA polymerase, to the coordinated repair of “dirty” or damaged DNA ends that block BER. These scenarios become particularly biologically relevant during times where there is an increase in genome instability (i.e., in cancer cells and/or during therapeutic treatments). The overarching goal of this proposal is to understand the molecular mechanisms of each BER component individually and to place these activities within the larger BER co-complex with damaged DNA repair intermediates. Elegant biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for interpreting the biological response and the subsequent development of therapeutic treatments. We are in a unique position to advance this scientific front based on my strong track record in DNA damage and repair, assembled team of collaborators, and multidisciplinary approach. To meet this goal, we utilize a comprehensive approach of time-lapse X-ray crystallography, neutron crystallography, small angle neutron scattering, molecular dynamic simulations, enzyme kinetics, and single-molecule total internal reflection microscopy. Using these methodologies, we will determine 1) How the location of DNA damage alters the DNA polymerase mechanism during repair; 2) How does the poorly characterized APE1 exonuclease reaction process damaged RNA and DNA repair blocks; 3) What are the mechanistic roles of protons during DNA damage and repair; 4) How are DNA repair complexes formed and structurally organized? This set of questions will go from an atomic level mechanistic understanding of key BER components to the structural and dynamic interactions within the entire BER multi-protein complex. By doing this, we will lay the foundation to address an inherent challenge in establishing cellular models and developing new therapeutic treatments that target DNA repair. With this information in hand, we will be closer to our long-term goal of providing a basis for rational drug design towards the development of more effective chemotherapeutics and synergistic drug combinations that target proteins involved in the DNA damage response.

氧化应激是一种普遍且危险的细胞状况，导致对 DNA的结构。这些修改促进了诱变，因此众多 人类疾病，包括癌症。基本惊喜维修（BER）途径是电池的主要防御 氧化DNA损伤是基因组稳定性的重要监护人。而单个酶在 经典的BER周期在很大程度上建立了，这些酶如何在A中起作用 多蛋白/DNA复合物可促进每种蛋白质之间有毒DNA修复中间体的通道。 此外，众所周知，经典BER途径中的出发如何影响DNA修复过程 和基因组稳定性。这些不匹配，损坏，和核糖核苷酸插入的不匹配，损坏和核糖 - DNA聚合酶，对“脏”或受损的DNA的协调修复阻断了BER。这些场景 在基因组不稳定性增加的时候变得特别相关（即 癌细胞和/或治疗期间）。该提议的总体目标是了解 每个BER组件的分子机制单独地将这些活动放置在较大的BER中 具有损坏的DNA修复中间体的共复合。需要阐明优雅的生物物理方法 这些复杂性，既是解释生物学反应的基础，又为 随后的治疗疗法发展。我们处于一个独特的位置，可以推进这一科学阵线 根据我在DNA损坏和维修方面的良好记录，组装合作者团队，以及 多学科方法。为了实现这一目标，我们采用了延时X射线的全面方法 晶体学，中子晶体学，小角度散射，分子动态模拟， 酶动力学和单分子总内反射显微镜。使用这些方法，我们将 确定1）DNA损伤的位置如何改变修复过程中DNA聚合酶机制； 2）如何 APE1外切酶反应过程的表征较差是否损坏了RNA和DNA修复块； 3） 质子在DNA损伤和修复过程中的机械作用是什么？ 4）DNA修复复合物如何 形成和结构组织？这组问题将从原子水平的机械理解中 在整个BER多蛋白复合物中的结构和动态相互作用的关键BER组件的。 通过这样做，我们将奠定基础，以应对建立蜂窝模型和 开发针对DNA修复的新的治疗方法。有了这些信息，我们将更加接近 我们的长期目标是为发展更有效的发展提供理性药物设计的基础 靶向参与DNA损伤的蛋白质的化学治疗剂和协同药物组合 回复。