Structure and mechanism of CpG specific DNA glycosylases

描述（由申请人提供）：DNA编码的遗传信息的完整性对所有生物体都是必不可少的，但DNA的反应性碱基不断受到内源性和外源性来源的化学修饰。为了抵消这种不可避免的损害，细胞机制包括DNA修复系统。DNA中受损的碱基通过碱基切除修复（BER）修复，由损伤特异性DNA糖基化酶启动。这些酶在大量基因组DNA中发现损伤，并水解通常稳定的键以释放受损的碱基，产生脱嘌呤/脱嘧啶（AP）DNA。DNA糖基化酶通常与细胞毒性AP DNA产物紧密结合，直到被AP核酸内切酶取代以继续BER。虽然有些酶识别单个病变;例如，尿嘧啶DNA糖基化酶对尿嘧啶具有精确的特异性，其他糖基化酶识别多个损伤和/或错配碱基。两种人类酶识别G/T错配和其他诱变损伤，特别是在CpG位点;甲基结合结构域IV（MBD 4）和胸腺嘧啶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杂和多种形式的损伤，但排除正常的碱基对，它们如何催化通常稳定的键的水解，以及AP DNA产物如何从DNA糖基化酶转移到AP核酸内切酶。该建议的重点是确定TDG如何获得其对G/T错配和其他病变的特异性，以及其对正常GC对的特异性，以及人AP核酸内切酶（APE 1）如何刺激AP DNA从TDG中释放。为此，我们将采用多学科的方法，包括结构，生物物理和生物化学方法。具体目标是（i）确定TDG催化结构域（TDGc）的NMR结构，（ii）使用瞬态和稳态动力学以及平衡结合实验来表征TDG反应机理，（iii）发现TDG识别多个底物和排斥GC的化学基础，（iv）阐明TDG和APE 1之间的AP DNA转移机制，和（v）测定二元TDGc-DNA底物类似物复合物的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位点损伤的致突变和细胞毒性效应，TDG的拟议结构和机制研究可能对衰老和包括癌症在内的疾病具有重要意义。

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.