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。 此外，人们对经典BER通路中的偏差如何影响DNA修复过程知之甚少 和基因组的稳定性。这些偏差的范围从错配、损坏和核糖核酸插入 DNA聚合酶，以协调修复“脏的”或损坏的DNA末端，阻止BER。这些场景 在基因组不稳定性增加的时候变得特别具有生物学意义(即 癌细胞和/或治疗期间)。这项建议的首要目标是理解 每个误码率分量的分子机制，并将这些活动放置在较大的误码率中 与受损的DNA修复中间体形成共复合体。需要优雅的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 随后的治疗方法的发展。我们在推进这一科学战线方面处于独特的地位 基于我在DNA损伤和修复方面的良好记录，组建的合作者团队，以及 多学科方法。为了实现这一目标，我们使用了一种综合的延时X射线方法 结晶学、中子结晶学、小角中子散射、分子动力学模拟、 酶动力学和单分子全内反射显微镜。使用这些方法，我们将 确定1)DNA损伤的位置如何在修复过程中改变DNA聚合酶机制；2)如何 特征不佳的APE1核酸外切酶反应过程是否破坏了RNA和DNA修复块； 质子在DNA损伤和修复中的机制作用是什么；4)DNA修复复合体是如何 形成并有组织的吗？这组问题将从原子水平的机械论理解出发 在整个误码率多蛋白质复合体中，关键误码率成分的结构和动态相互作用。 通过这样做，我们将为解决建立细胞模型和 开发针对DNA修复的新治疗方法。有了这些信息，我们将更接近 我们的长期目标是为合理的药物设计提供基础，朝着更有效的方向发展 针对参与DNA损伤的蛋白质的化疗药物和协同药物组合 回应。

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.