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，会导致细胞发生有害改变。 DNA 的结构。这些修饰促进诱变，从而促进许多人类疾病，包括癌症。碱基切除修复 (BER) 途径是细胞的主要防御机制 DNA 氧化损伤是基因组稳定性的重要守护者。虽然单个酶在过程中的作用经典的 BER 循环已基本建立，但这些酶如何在一个循环中一起发挥作用仍然是个谜。多蛋白质/DNA 复合物，促进有毒 DNA 修复中间体在每种蛋白质之间的通道。重要的是，BER 不仅发生在裸露的双链 DNA 上，而且也发生在由以下物质组成的染色质内：核小体。这些核小体对 BER 酶访问和有效修复 DNA 构成了障碍损害。 DNA 修复蛋白克服这一障碍以修复 DNA 损伤的机制对核小体了解甚少。该提案的主要目标是了解分子机制使用裸双链 DNA 单独和在更大的多蛋白/DNA 复合物中分析每个 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反应和治疗方法的发展。我们处于独特的地位基于我在 DNA 损伤和修复方面的良好记录，为了推进这一科学前沿，组建了以下团队：合作者和多学科方法。为了实现这一目标，我们采用了一种综合的时间方法延时X射线晶体学、分子动力学模拟、酶动力学、单分子全内部反射显微镜和冷冻电镜。使用这些方法，我们将确定 1) 新的基本面如何机械步骤改变 DNA 聚合酶和端粒酶机制； 2) 单个 BER 酶如何组装成多蛋白质/DNA复合物，以促进有毒DNA中间体的通道； 3）怎么样多蛋白/DNA BER 复合体结构组织良好； 4）DNA损伤是如何识别和修复的核小体； 5) 多蛋白/DNA BER 复合物是如何在含有 DNA 的核小体上形成的损害。这组问题将从对关键 BER 组件的原子级机械理解出发整个 BER 多蛋白复合物内的结构和动态相互作用。通过这样做，我们将奠定为解决建立细胞模型和开发新疗法的固有挑战奠定了基础针对 DNA 修复的治疗。有了这些信息，我们将更接近我们的长期目标为合理的药物设计提供基础，以开发更有效的化疗药物针对参与 DNA 损伤反应的蛋白质的协同药物组合。