Regulations of DNA Alkylation/Deamination Damage Repair

DNA烷基化/脱氨基损伤修复的调控

• 批准号: 6865662
• 负责人: RABINDRA ROY
• 金额: $ 27.63万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865662
• 关键词: DNA damage; DNA repair; N glycosidase; alkylation; amination; endonuclease; enzyme activity; enzyme mechanism; enzyme substrate complex; posttranslational modifications; protein protein interaction

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.

描述（由申请人提供）：内源和外源产生的反应性氮种和烷基化剂是细胞遗传毒性的主要原因，这被认为会导致多种疾病，例如癌症和衰老。这种烷基化和脱氧的碱病变通常通过碱基切除修复（BER）途径进行修复，当DNA糖基化酶去除受损的碱时，该途径启动。其中，N-甲基嘌呤DNA-糖基化酶（MPG）修复了一系列结构上不同的嘌呤，这些葡萄糖酶（MPG）都存在于从细菌到人的所有物种中。尽管最近通过X射线晶体学来解决了人类（H）MPG的3D结构，但对于识别具有各种结构的底物或随后催化的底物的详细机制知之甚少。我们的初步结果表明，AP-核酸酶与MPG相互作用并刺激。此外，我们的新型初步观察表明，MPG是长斑块Ber复合物的一个组成部分，为我们的中心假设提供了基础工作，即与细胞因子（包括其他BER蛋白或翻译后修改）的相互作用可以选择BER子路口（短或长斑块）（短或长斑块），并调节MPG的活性，以使MPG的活性近似其细胞的催化速率，并依靠其相反的情况。 我们的具体目的是：（1）通过隔离一系列位点特异性MPG突变体并测试其在非目标，不匹配的，底物DNA结合和弯曲以及基础底线和基础底漆和基础底漆和基础分析中，确定MPG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； （3）通过开发基于新型的基于基于体内的质粒的新质粒，阐明了烷基化和脱氨基碱基的短和长底基切除修复途径的体内状态。 我们的长期目标是对MPG的作用和调节的全面了解，这是哺乳动物BER系统修复细胞烷基化/脱氨酸损伤的组成部分。这些知识将使我们最终制定策略来调节MPG表达以进行化学预防和治疗目的。