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(申请人提供)：遗传病和癌症中很大比例的突变是CpG位点的CAET过渡，主要由5-甲基胞嘧啶(M5C)脱氨基产生，从而产生G.T错配。CpG位点上的胞嘧啶甲基化是转录沉默的标志，对许多细胞过程至关重要，对胚胎发育也是必不可少的。因此，保持CpG的完整性对于避免突变和适当的转录调控是重要的。识别G.T损伤的两种DNA糖基酶是胸腺嘧啶DNA糖基酶(TDG)和甲基结合结构域IV(MBD4)。这些糖基酶启动碱基切除修复(BER)途径，将靶核苷酸(DT)翻转到它们的活性部位，并裂解碱糖(N-糖基)键，产生一个碱性(AP)位点。AP内切酶(APE1)和下游的BER酶的修复继续进行。TDG和MBD4面临着从不匹配的配对中移除正常碱基的艰巨任务，必须在有效的G.T修复和避免未损坏的DNA之间取得平衡，这可能会限制它们的活性。鉴于为了避免突变和转录调控而保持CpG完整性的生物学需要，重要的是要详细了解TDG和MBD4是如何识别和移除病变的，为什么它们对G.T错对的活性很慢(这可能会影响CpG的可变性)，以及APE1是如何调节它们的活性的。为此，我们提出了生化、生物物理和结构方法的强大组合，以实现四个特定目标：(I)我们将使用瞬时动力学和平衡结合方法来确定控制TDG和MBD4的病变识别、病变切除和产品释放的参数，揭示为什么它们的周转对于G.T病变来说是缓慢的，而对于5-卤代尿嘧啶(如5FU)的切除是快速的。(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CpG位点，但不作用于CpG位点，我们将尝试解决与CpG DNA结合的TDG的结构。(Iii)我们将使用瞬时动力学和核磁共振方法，结合稳定的TDG-AP-DNA复合体，揭示APE1如何调节TDG活性(提高其周转)。我们还将检验这一假设，即TDG的SUMO化是及时释放产品和有效修复G.T损伤所必需的。(Iv)我们将使用核磁共振来确定TDG内在无序的N-末端区域如何使G.T修复有效，以及无序的C-末端区域如何与相扑蛋白质形成非共价相互作用，这对于相扑调节TDG活性和TDG与相扑修饰蛋白(即p731，PML)的结合是重要的。了解内在无序蛋白质区域的功能是结构生物学的一个主要挑战，我们的研究将为这一新兴领域做出贡献。这些研究的成功完成将推动我们理解突变的G.T损伤是如何在人类中识别和修复的，为什么CpG位点是突变热点，以及TDG如何介导广泛使用的抗癌药物5FU的细胞毒性，并可能对TDG和MBD4在癌症、遗传病和5FU化疗中的作用产生影响。