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: 8535460
• 负责人: Alex C Drohat
• 金额: $ 15.54万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535460
• 关键词: Active Sites; Address; Affinity; Antineoplastic Agents; Base Excision Repairs; Base Pairing; Binding; Biochemical; Biological; C-terminal; Catalytic Domain; Cell physiology; Cleaved cell; Complex; Cytosine; DNA; DNA Repair Enzymes; DNA glycosylase; DNA-(apurinic or apyrimidinic site) lyase; Deamination; Disease; Disulfides; E1A-associated p300 protein; Embryonic Development; Environment; Equilibrium; Excision; Face; Fluorouracil; Frequencies; Future; Gene Mutation; Hereditary Disease; Human; Kinetics; Lesion; Malignant Neoplasms; Mediating; Methods; Methylation; Modeling; Mutation; N-terminal; Nucleotides; Pathway interactions; Process; Protein Region; Proteins; Regulation; Role; Site; Small Ubiquitin-Related Modifier Proteins; Specificity; Structure; Testing; Thymine DNA Glycosylase; Transcriptional Regulation; Uracil; Work; analog; base; cancer genetics; chemotherapy; crosslink; cytotoxicity; demethylation; human APEX1 protein; human DNA; human disease; protein function; protein p73; repair enzyme; repaired; scaffold; structural biology; sugar

DESCRIPTION (provided by applicant): A large percentage of mutations in genetic disease and cancer are CAET transitions at CpG sites, generated mainly by deamination of 5-methylcytosine (m5C) to give G.T mispairs. Cytosine methylation at CpG sites is a mark for transcriptional silencing that is central to many cellular processes and essential for embryogenesis. Thus, maintaining CpG integrity is important for mutation avoidance and proper transcriptional regulation. Two DNA glycosylases recognize G.T lesions, thymine DNA glycosylase (TDG) and methyl binding domain IV (MBD4). Initiating the base excision repair (BER) pathway, these glycosylases flip the target nucleotide (dT) into their active site and cleave the base-sugar (N-glycosylic) bond, producing an abasic (AP) site. Repair continues with AP endonuclease (APE1) and downstream BER enzymes. TDG and MBD4 face the daunting task of removing a normal base from a mismatched pair, and must balance the needs for efficient G.T repair and avoidance of undamaged DNA, which may limit their activity. Given the biological need to maintain CpG integrity for mutation avoidance and transcriptional regulation, it is important to obtain a detailed understanding of how TDG and MBD4 recognize and remove lesions, why their activity is slow for G.T mispairs (which may impact CpG mutability) and how their activity is regulated by APE1. Towards this end, we propose a powerful combination of biochemical, biophysical, and structural methods to achieve four specific aims: (i) We will use transient kinetics and equilibrium binding methods to determine the parameters that govern lesion recognition, lesion excision, and product release for TDG and MBD4, revealing why their turnover is slow for G.T lesions and fast for excision of 5-halogenated uracils such as 5FU. (ii) We will determine the crystal structure of TDG (catalytic domain) bound to DNA containing a non-cleavable substrate analog, revealing interactions that promote G.T specificity. To understand how TDG interrogates but does not act upon CpG sites, we will attempt to solve the structure of TDG bound to CpG DNA. (iii) We will use transient kinetics, and NMR methods with a stable TDG-AP-DNA complex, to reveal how APE1 regulates TDG activity (enhances its turnover). We will also test the hypothesis that SUMOylation of TDG is required for timely product release and efficient repair of G.T lesions. (iv) We will use NMR to determine how the intrinsically disordered N-terminal region of TDG enables efficient G.T repair, and how the disordered C-terminal region forms non-covalent interactions with SUMO proteins, which is important for SUMO regulation of TDG activity and TDG binding to SUMO-modified proteins (i.e., p731, PML). Understanding the function of intrinsically disordered protein regions is a major challenge in structural biology, and our studies will contribute to this emerging field. Successful completion of these studies will advance our understanding of how mutagenic G.T lesions are recognized and repaired in humans, why CpG sites are mutational hotspots, and how TDG mediates the cytotoxicity of 5FU, a widely used anti-cancer drug, with implications for the role of TDG and MBD4 in cancer, genetic disease, and 5FU chemotherapy.

描述（由申请人提供）：遗传疾病和癌症中的大部分突变是 CpG 位点的 CAET 转变，主要是通过 5-甲基胞嘧啶 (m5C) 脱氨基作用产生 G.T 错配。 CpG 位点的胞嘧啶甲基化是转录沉默的标志，它是许多细胞过程的核心，并且对于胚胎发生至关重要。因此，维持 CpG 完整性对于避免突变和适当的转录调控非常重要。两种 DNA 糖基化酶可识别 G.T 损伤：胸腺嘧啶 DNA 糖基化酶 (TDG) 和甲基结合域 IV (MBD4)。这些糖基化酶启动碱基切除修复 (BER) 途径，将目标核苷酸 (dT) 翻转到其活性位点并裂解碱基糖（N-糖基）键，产生脱碱基 (AP) 位点。 AP 核酸内切酶 (APE1) 和下游 BER 酶继续进行修复。 TDG 和 MBD4 面临着从不匹配的碱基对中去除正常碱基的艰巨任务，并且必须平衡有效 G.T 修复的需求和避免未损坏的 DNA，这可能会限制它们的活性。鉴于维持 CpG 完整性以避免突变和转录调控的生物学需要，重要的是详细了解 TDG 和 MBD4 如何识别和消除病变、为什么它们的活性对于 G.T 错配缓慢（这可能会影响 CpG 突变性）以及如何它们的活性受 APE1 调节。为此，我们提出了生物化学、生物物理和结构方法的强大组合，以实现四个具体目标：（i）我们将使用瞬态动力学和平衡结合方法来确定控制病变识别、病变切除和产物释放的参数对于 TDG 和 MBD4，揭示了为什么它们对于 GT 病变的周转速度较慢，而对于 5-卤代尿嘧啶（如 5FU）的切除则周转速度较快。 (ii) 我们将确定与含有不可切割底物类似物的 DNA 结合的 TDG（催化结构域）的晶体结构，揭示促进 G.T 特异性的相互作用。为了了解 TDG 如何询问但不作用于 CpG 位点，我们将尝试解析与 CpG DNA 结合的 TDG 的结构。 (iii) 我们将使用瞬态动力学和 NMR 方法以及稳定的 TDG-AP-DNA 复合物来揭示 APE1 如何调节 TDG 活性（增强其周转率）。我们还将检验这样的假设：TDG 的 SUMO 化对于产品的及时发布和 GT 损伤的有效修复是必需的。 (iv) 我们将使用 NMR 来确定 TDG 本质上无序的 N 端区域如何实现有效的 G.T 修复，以及无序的 C 端区域如何与 SUMO 蛋白形成非共价相互作用，这对于 TDG 活性的 SUMO 调节非常重要TDG 与 SUMO 修饰蛋白（即 p731、PML）结合。了解本质上无序的蛋白质区域的功能是结构生物学的一个重大挑战，我们的研究将为这个新兴领域做出贡献。这些研究的成功完成将加深我们对人类突变性 G.T 病变如何识别和修复、为什么 CpG 位点是突变热点以及 TDG 如何介导 5FU（一种广泛使用的抗癌药物）的细胞毒性的理解，并对其作用产生影响。 TDG 和 MBD4 在癌症、遗传病和 5FU 化疗中的作用。