DNA-Protein Dynamics in Base Excision DNA Repair

描述（由申请人提供）： DNA碱基的自发损伤是遗传不稳定性的主要来源。未能纠正这种损伤会导致体细胞突变，从而导致许多与衰老相关的疾病。防止这些自发性病变的能力主要依赖于碱基切除修复（BER）途径，其中DNA糖基化酶扫描基因组以定位和切除碱基病变。BER通路中涉及的许多常见形式的损伤和蛋白质已经被鉴定和结构表征，但我们对这些酶如何完成全基因组修复这一惊人任务的理解存在根本性的差距。在这一空白被填补之前，DNA损伤和诱变的研究将主要是观察性的，我们将无法预测多态性或暴露于新致癌物的影响。我们的长期目标是了解BER蛋白定位和选择性作用于基因组DNA内广泛的DNA损伤的分子机制和基本原理。核心假设，即蛋白质-DNA动力学对BER途径的功能至关重要，特别是对那些识别多种形式DNA损伤的酶，得到了我们实验室和该领域其他工作人员的数据的有力支持。在过去的研究进展和初步数据的指导下，我们提出了四个具体目标：1）确定人DNA糖基化酶的体外搜索机制; 2）评估促进扩散有助于真核细胞中有效DNA修复的假设; 3）通过使用各种结构多样的碱基损伤的竞争动力学来量化AAG的催化特异性; 4）确定AAG实现广泛底物的有效识别的分子机制。通过结合稳态前酶动力学、荧光光谱和结构-活性关系的结果，我们准备剖析对于识别和修复受损碱基重要的蛋白质-DNA动力学。这种方法是创新的，因为我们正在开发新的生化分析和新的分子模型，以了解DNA修复的动力学和热力学机制。这项研究意义重大，因为它很有可能扩大我们对BER机制的理解，并揭示与其他DNA模板生物过程相关的基本原理。由于BER是细胞防御基因组不稳定性（包括内源性损伤）的关键组成部分，因此这些研究将有助于我们理解诱变和正常衰老过程。

DESCRIPTION (provided by applicant): Spontaneous damage of DNA bases is a major source of genetic instability. Failure to correct this damage leads to somatic mutations that underlie many diseases associated with aging. The ability to safeguard against these spontaneous lesions relies largely on the base excision repair (BER) pathway whereby DNA glycosylases scan the genome to locate and excise base lesions. Many common forms of damage and proteins involved in the BER pathway have been identified and structurally characterized, but there is a fundamental gap in our understanding of how these enzymes accomplish this amazing task of genome-wide repair. Until this gap is filled, studies of DNA damage and mutagenesis will be largely observational and we will not be able to predict the effects of polymorphisms or exposure to novel carcinogens. Our long-term goal is to understand the molecular mechanisms and fundamental principles by which BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA. The central hypothesis, that protein-DNA dynamics are critical to the function of the BER pathway, particularly to those enzymes that recognize multiple forms of DNA damage, is strongly supported by data from our lab and others working in this area. Guided by progress and preliminary data from the past funding period, we propose to pursue four specific aims: 1) Determine the searching mechanism(s) of human DNA glycosylases in vitro; 2) Evaluate the hypothesis that facilitated diffusion contributes to efficient DNA repair in eukaryotic cells; 3) Quantify the catalytic specificity of AAG by employing competition kinetics with a wide variety of structurally diverse base lesions; 4) Determine the molecular mechanisms by which AAG achieves efficient recognition of a broad range of substrates. By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we are poised to dissect the protein-DNA dynamics important for recognition and repair of damaged bases. The approach is innovative, because we are developing novel biochemical assays and new molecular models to understand the kinetic and thermodynamic mechanisms of DNA repair. The proposed research is significant, because it has a high probability of expanding our understanding of BER mechanisms and to uncovering fundamental principles that are relevant for other DNA-templated biological processes. As BER is a critical component of the cellular defense against genomic instability, including endogenous damage, these studies will contribute both to our understanding of mutagenesis and the normal aging process.