Structural and Mechanistic Studies of DNA Repair

氧化应激是一种普遍而危险的细胞疾病，会导致有害的 DNA的结构。这些修饰促进了突变，从而发展了许多 人类的疾病，包括癌症。碱基切除修复(BER)途径是细胞对 氧化DNA损伤，是基因组稳定性的重要守护者。而单个酶在生物合成过程中的作用 经典的误码率循环在很大程度上已经建立，但这些酶是如何在一个 多蛋白质/DNA复合体，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，误码不仅发生在裸露的双链DNA上，而且也发生在由以下成分组成的染色质中 核小体。这些核小体是BER酶访问和有效修复DNA的障碍 损坏。DNA修复蛋白克服这一障碍修复DNA损伤的机制 人们对核小体知之甚少。这项提议的主要目标是了解分子机制。 使用裸露双链DNA分别和较大的多蛋白质/DNA复合体中的每个误码率因子 和染色质；并破译端粒酶复制端粒的分子机制。雅致 需要生物物理方法来阐明这些误码率的复杂性，并为 解释生物反应和治疗方法的发展。我们处于一个独特的位置 基于我在DNA损伤和修复方面的出色记录，为了推进这一科学前沿， 合作者和多学科方法。为了实现这一目标，我们利用了综合的时间方法- 失活X射线结晶学、分子动力学模拟、酶动力学、单分子总内部 反射显微镜和低温电子显微镜。使用这些方法，我们将确定1)新的基础 机械性步骤改变DNA聚合酶和端粒酶机制；2)单个BER酶是如何 组装成多蛋白质/DNA复合体，以促进有毒DNA中间体的运输；3)如何 多蛋白质/DNA BER复合体的结构组成；4)DNA损伤是如何在 以及5)含有DNA的核小体上如何形成多蛋白质/DNA BER复合体 损坏。这组问题将从原子级别对关键误码率组件的机械理解出发 到整个BER多蛋白质复合体内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法方面解决固有挑战的基础 以DNA修复为目标的治疗。有了这些信息，我们将更接近我们的长期目标 为合理设计药物以开发更有效的化疗药物和 以参与DNA损伤反应的蛋白质为靶点的协同药物组合。

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.