Regulations of DNA Alkylation/Deamination Damage Repair

描述（由申请人提供）：内源性和外源性产生的活性氮物质和烷化剂是细胞遗传毒性的主要原因，被认为会导致多种疾病，如癌症和衰老。这种烷基化和脱氨基的碱基损伤通常通过碱基切除修复（BER）途径修复，当DNA糖基化酶去除受损的碱基时启动。其中，一系列结构不同的受损嘌呤被修复的N-甲基嘌呤DNA-糖基化酶（MPG），存在于所有物种从细菌到man. Although人（h）MPG的3D结构最近已解决了X射线晶体学，很少知道其识别具有不同结构的底物的详细机制，或随后的催化。我们的初步结果表明，AP-内切酶与MPG相互作用，并刺激MPG。此外，我们的新的初步观察表明，MPG是长补丁BER复合物的一个组成部分提供了基础工作，以测试我们的中心假设，即与细胞因子，包括其他BER蛋白或翻译后修饰的相互作用可能会选择BER子途径（短或长补丁）和调节MPG的活性，以接近其细菌对应物的催化速率取决于细胞的必要性。 我们的具体目标是：（1）通过分离一系列位点特异性MPG突变体并测试其在非靶、错配、底物DNA结合和弯曲以及碱基翻转和催化中的活性来确定MPG对底物识别和催化的分子机制;（2）通过测试蛋白质-蛋白质相互作用和详细的动力学分析来阐明BER或其他细胞蛋白对MPG酶活性的影响;和（3）通过开发基于新质粒的体内测定法阐明烷基化和脱氨基碱基的短和长补丁碱基切除修复途径的体内状态。 我们的长期目标是全面了解MPG作为哺乳动物BER系统的一个组成部分在修复细胞烷基化/脱氨损伤中的作用和调控。这些知识将使我们最终设计出用于化学预防和治疗目的的调节MPG表达的策略。

DESCRIPTION (provided by applicant): The reactive nitrogen species and alkylating agents generated endogenously and exogenously are the major cause of cellular genotoxicity, which is thought to cause multiple diseases such as cancer and aging. Such alkylated and deaminated base lesions are generally repaired via the base excision repair (BER) pathway, initiated when a DNA glycosylase removes the damaged base. Among these, a series of structurally diverse damaged purines are repaired by N-methylpurine DNA-glycosylase (MPG), present in all species from bacteria to man. Although 3D structure of human(h) MPG has recently been solved by X-ray crystallography, little is known about its detailed mechanisms for recognition of substrates with various structures, or for subsequent catalysis. Our preliminary results show that AP-endonuclease interacts with and stimulates MPG. Moreover, our novel preliminary observations suggesting that MPG is a component of long patch BER complex provide the ground work to test our central hypothesis that the interaction with cellular factors including other BER proteins or post-translational modification may select the BER subpathways (short or long patch) and modulate the activity of MPG to approximate the catalytic rates of its bacterial counterpart depending on cellular necessity. Our specific aims are to: (1) determine the molecular mechanisms of substrate recognition and catalysis by MPG by isolating a series of site-specific MPG mutants and testing for their activities in non-target, mismatched, substrate DNA binding and bending as well as base-flipping and catalysis; (2) elucidate the effect of BER or other cellular proteins on the enzymatic activity of MPG by testing protein-protein interactions and detailed kinetic analysis; and (3) elucidate the in vivo status of short and long patch base excision repair pathways of alkylated and deaminated bases by developing a novel plasmid based in vivo assay. Our long-term goal is comprehensive understanding of the role and regulation of MPG as a component of mammalian BER system for repair of cellular alkylation/deamination damage. This knowledge will allow us eventually to devise strategies for modulating MPG expression for chemopreventive and therapeutic purposes.