Structural and Mechanistic Studies of DNA Repair

DNA修复的结构和机制研究

基本信息

批准号：
10622967
负责人：
Bret D Freudenthal
金额：
$ 46.5万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2028-08-31
项目状态：
未结题

项目摘要

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. Importantly, BER not only occurs on naked duplex DNA, but also within chromatin that is composed of nucleosomes. These nucleosomes present a barrier to BER enzymes accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome is poorly understood. The major goals of this proposal are to understand the molecular mechanisms of each BER factor both individually and within larger multi-protein/DNA complexes using naked duplex DNA and chromatin; and to decipher the molecular mechanism by which telomerase replicates the telomere. Elegant biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for interpreting the biological response and the 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, molecular dynamic simulations, enzyme kinetics, single-molecule total internal reflection microscopy, and cryo-EM. Using these methodologies, we will determine 1) how do new fundamental mechanistic steps alter the DNA polymerase and telomerase mechanism; 2) how do individual BER enzymes assemble into a multi-protein/DNA complex to facilitate the channeling of toxic DNA intermediates; 3) how are multi-protein/DNA BER complexes structurally organized; 4) how is DNA damage identified and repaired within nucleosomes; and 5) how are multi-protein/DNA BER complexes formed on nucleosomes containing DNA damage. 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。基础切除修复（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。重要的是，BER不仅发生在裸体双链DNA上，而且在染色质中也发生核小组。这些核小体呈现出可进入并有效修复DNA的BER酶的障碍损害。 DNA修复蛋白克服这种障碍以修复DNA损伤的机制核小体了解不足。该提案的主要目标是了解分子机制使用裸式双工DNA单独和较大的多蛋白/DNA复合物中的每个BER因子和染色质；并破译端粒酶复制端粒的分子机制。优雅的需要生物物理方法来阐明这些BER复杂性，并为解释生物学反应和治疗治疗的发展。我们处于独特的位置为了根据我在DNA损坏和维修方面的良好记录来推进这一科学阵线，组装了团队合作者和多学科方法。为了实现这一目标，我们采用了全面的时间方法 - 失去X射线晶体学，分子动力学模拟，酶动力学，单分子总内部反射显微镜和冷冻EM。使用这些方法，我们将确定1）如何新基本机械步骤改变了DNA聚合酶和端粒酶机制； 2）单个BER酶如何组装成多蛋白/DNA复合物，以促进有毒DNA中间体的传播； 3）如何多蛋白/DNA BER络合物结构组织了； 4）如何在内部识别和修复DNA损伤核小体； 5）如何在含有DNA的核小体上形成多蛋白/DNA BER复合物损害。这组问题将从对关键BER组件的原子级别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。通过这样做，我们将在建立细胞模型和开发新的治疗方面应对固有挑战的基础靶向DNA修复的治疗方法。有了这些信息，我们将更接近我们的长期目标为开发更有效的化学治疗剂和靶向参与DNA损伤反应的蛋白质的协同药物组合。