Structure and mechanism of CpG specific DNA glycosylases

CpG特异性DNA糖基化酶的结构和机制

• 批准号: 6856982
• 负责人: Alex C Drohat
• 金额: $ 2万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6856982
• 关键词: CpG islands; DNA damage; DNA repair; N glycosidase; catalyst; chemical kinetics; endonuclease; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; hydrolysis; intermolecular interaction; ionic bond; nuclear magnetic resonance spectroscopy; protein binding; protein structure function; site directed mutagenesis; thermodynamics; thymine

DESCRIPTION (provided by applicant): The integrity of the genetic information encoded by DNA is essential to all organisms, yet the reactive bases of DNA are continuously subjected to chemical modification from endogenous and exogenous sources. To counteract this inevitable damage, the cellular machinery includes DNA repair systems. Damaged bases in DNA are repaired via base excision repair (BER), initiated by a damage-specific DNA glycosylase. These enzymes find lesions within the vast genomic DNA, and hydrolyze a generally stable bond to release the damaged base, producing apurinic/apyrimidinic (AP) DNA. DNA glycosylases typically bind the cytotoxic AP DNA product tightly until displaced by an AP endonuclease to continue BER. Although some enzymes recognize a single lesion; e.g., uracil DNA glycosylase is exquisitely specific for uracil, others recognize multiple lesions and/or mismatched bases. Two human enzymes recognize G/T mispairs, and other mutagenic lesions, specifically at CpG sites; methyl-binding domain IV (MBD4) and thymine DNA glycosylase (TDG). The long-term goal of this research is to understand how these enzymes recognize complex and multiple forms of damage and yet exclude normal base pairs, how they catalyze the hydrolysis of a generally stable bond, and how the AP DNA product is transferred from the DNA glycosylase to the AP endonuclease. The focus of this proposal is to determine how TDG obtains its specificity for G/T mispairs and other lesions, and its specificity against normal GC pairs, and how human AP endonuclease (APE1) stimulates the release of AP DNA from TDG. Towards this end, we will employ a multidisciplinary approach including structural, biophysical, and biochemical methods. The specific aims are to (i) determine the NMR structure of the TDG catalytic domain (TDGc), (ii) characterize the TDG reaction mechanism using transient and steady-state kinetics, and equilibrium binding experiments, (iii) discover the chemical basis of the recognition of multiple substrates and the rejection of GC by TDG, (iv) elucidate the mechanism of AP DNA transfer between TDG and APE1, and (v) determine the NMR structure of a binary TDGc-DNA substrate analog complex to reveal the structural basis of TDG specificity. Given the mutagenic and cytotoxic effects of damage occurring at CpG sites in human genomic DNA, the proposed structural and mechanistic studies of TDG may have significant implications for ageing, and diseases including cancer.

描述（由申请人提供）：DNA编码的遗传信息的完整性对于所有生物都是必不可少的，但是DNA的反应性碱不断受到内源和外源性来源的化学修饰。为了抵消这种不可避免的损害，细胞机械包括DNA修复系统。通过碱基切除修复（BER）修复DNA中受损的碱基，该修复由损伤特异性DNA糖基化酶引发。这些酶在巨大的基因组DNA中找到病变，并水解通常稳定的键以释放受损的碱，从而产生肾上腺素/阿哌替氨酸（AP）DNA。 DNA糖基酶通常紧密地结合细胞毒性AP DNA产物，直到被AP内切核酸酶移动以继续ber。尽管有些酶识别出一个病变。例如，尿嘧啶DNA糖基化酶是尿嘧啶的精确特异性，其他人则识别多个病变和/或不匹配的碱。两种人类酶识别g/t的误解和其他诱变病变，特别是在CPG部位。甲基结合结构域IV（MBD4）和胸腺胺DNA糖基化酶（TDG）。这项研究的长期目标是了解这些酶如何识别复合物和多种形式的损伤，但排除了正常的碱基对，如何催化通常稳定键的水解以及如何将AP DNA产物从DNA糖基酶转移到AP核酸内核酸酯。该提案的重点是确定TDG如何获得其对G/T误以和其他病变的特异性及其针对正常GC对的特异性，以及人类AP核酸内核酸酶（APE1）如何刺激从TDG中释放AP DNA。为此，我们将采用一种多学科方法，包括结构，生物物理和生化方法。 The specific aims are to (i) determine the NMR structure of the TDG catalytic domain (TDGc), (ii) characterize the TDG reaction mechanism using transient and steady-state kinetics, and equilibrium binding experiments, (iii) discover the chemical basis of the recognition of multiple substrates and the rejection of GC by TDG, (iv) elucidate the mechanism of AP DNA transfer between TDG and APE1和（V）确定二元TDGC-DNA底物模拟复合物的NMR结构，以揭示TDG特异性的结构基础。鉴于人类基因组DNA中CPG部位发生的损伤的诱变和细胞毒性作用，TDG的拟议的结构和机械研究可能对衰老以及包括癌症在内的疾病具有重要意义。