Protein-DNA Dynamics in Base Excision DNA Repair

描述(申请人提供)：DNA碱基的自发损伤是致癌突变的主要来源。鉴于每个基因组每天产生数以千计的病变，值得注意的是，癌症仍然是一种相对罕见的事件，大多数病例发生在生命相对较晚的时候。随着预期寿命的延长和接触外源性DNA损伤剂，社会承担着诊断和治疗癌症的不断增加的成本。在细胞水平上，抵御这些自发损害的能力在很大程度上依赖于碱基切除修复(BER)途径，DNA糖基酶通过该途径扫描基因组来定位和切除碱基损害。脱氧核糖核酸(AP)特异性内切酶、AP-裂解酶/DNA聚合酶和DNA连接酶的作用是完成DNA修复所必需的。我们的长期目标是了解BER蛋白如何定位并选择性地作用于基因组DNA中的广泛DNA损伤，以及蛋白质-蛋白质和蛋白质-DNA相互作用的动态如何使多步骤、多酶修复路径的协调。最近的证据表明，核苷酸翻转，即从DNA双链中提取核苷酸并结合到活性部位口袋中的过程，在区分受损和未受损碱基方面提供了很大的选择性。我们建议通过直接观察DNA糖基酶对受损和未受损核苷酸的翻转来检验这一假设(目标1)。通过检查每个DNA结合事件的许多核苷酸，促进了对罕见病变的基因组搜索，因此我们将表征BER酶沿DNA移动的能力，并测量在扫描过程中有效参与损伤位置的效率(目标2)。由于DNA修复中间体具有潜在的细胞毒性或致突变性，因此启动BER事件的完成至关重要。我们建议研究BER中蛋白质-蛋白质相互作用的动力学，并确定它们在协调多种酶活性方面的功能意义(目标3)。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，我们有一个独特的机会来剖析对损伤识别和修复至关重要的蛋白质-DNA动力学。由于BER是抗癌细胞防御的关键组成部分，而且这些通路对某些用于癌症治疗的DNA损伤剂具有拮抗作用，这些研究有可能有助于我们对突变的理解和癌症治疗的进展。

DESCRIPTION (provided by applicant): Spontaneous damage of DNA bases is a major source of cancer-causing mutations. Given the thousands of lesions generated per genome every day, it is remarkable that cancer remains a relatively infrequent event with the majority of cases arising relatively late in life. With increasing life expectancy and exposure to exogenous DNA damaging agents, society bears the ever increasing cost of diagnosing and treating cancer. At the cellular level 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. The action of an apurinic (AP)-specific endonuclease, AP-lyase/DNA polymerase, and DNA ligase are required to complete repair of the DNA. Our long-term goals are to understand how BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA and how the dynamics of protein-protein and protein-DNA interactions enable coordination of multi-step, multi-enzyme repair pathways. Recent evidence suggests that nucleotide flipping, the process by which a nucleotide is extracted from the DNA duplex and bound in an active site pocket, provides much of the selectivity in distinguishing damaged and undamaged bases. We propose to test this hypothesis by directly observing flipping of damaged and undamaged nucleotides by DNA glycosylases (Aim 1). The genomic search for rare lesions is facilitated by the examination of many nucleotides with each DNA binding event, therefore we will characterize the ability of BER enzymes to move along DNA and measure the efficiency with which sites of damage are productively engaged during a scanning encounter (Aim 2). As DNA repair intermediates are potentially cytotoxic or mutagenic, it is critical that initiated BER events be completed. We propose to investigate the dynamics of protein-protein interactions in BER and determine their functional significance in the coordination of multiple enzymatic activities (Aim 3). By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we have a unique opportunity to dissect the protein-DNA dynamics important for damage recognition and repair. As BER is a critical component of the cellular defense against cancer, and because these pathways are antagonistic toward some DNA damaging agents used in the treatment of cancer, these studies have the potential to contribute both to our understanding of mutagenesis and to advances in cancer therapy.