Regulations of DNA Alkylation/Deamination Damage Repair

描述(申请人提供)：内源和外源产生的活性氮物种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。这种烷基化和脱氨的碱基损伤通常通过碱基切除修复(BER)途径修复，该途径是当DNA糖基酶移除受损碱基时启动的。其中，一系列结构不同的损伤的嘌呤由N-甲基嘌呤DNA糖基酶(MPG)修复，从细菌到人类的所有物种中都存在这种酶。虽然最近X射线结晶学已经解决了人(H)MPG的三维结构，但对其识别各种结构的底物或随后的催化作用的详细机制还知之甚少。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，我们新的初步观察表明MPG是长斑块BER复合体的组成部分，这为验证我们的中心假设提供了基础，即与包括其他BER蛋白在内的细胞因子的相互作用或翻译后修饰可能选择BER亚途径(短或长斑块)，并调节MPG的活性，以根据细胞的需要近似其细菌对应物的催化速率。 我们的具体目标是：(1)通过分离一系列位点特异的MPG突变体并测试它们在非靶标、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识别和催化的分子机制；(2)通过测试蛋白质-蛋白质的相互作用和详细的动力学分析，阐明BER或其他细胞蛋白对MPG酶活性的影响；以及(3)通过开发一种基于体内实验的新型质粒基，阐明烷化和脱氨碱基的短和长片段碱基切除修复途径在体内的状况。 我们的长期目标是全面了解MPG作为哺乳动物BER系统的组成部分在修复细胞烷基化/脱氨损伤中的作用和调节。这一知识将使我们最终能够设计出调控MPG表达的策略，用于化学预防和治疗目的。

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.