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）：遗传疾病和癌症中很大一部分的突变是CPG部位的CAET转变，主要是通过5-甲基霉菌素（M5C）脱氨基来产生的，从而给予G.T mistairs。 CPG部位的胞嘧啶甲基化是转录沉默的标志，这对于许多细胞过程至关重要，对于胚胎发生至关重要。因此，保持CpG完整性对于避免突变和适当的转录调控很重要。两种DNA糖基酶识别G.T病变，胸腺胺DNA糖基化酶（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）翻转到其活性位点，并裂解碱基（N-聚糖）键，从而产生一个abasic（AP）位点。维修继续使用AP核酸内切酶（APE1）和下游BER酶。 TDG和MBD4面临着从不匹配的一对去除正常基础的艰巨任务，并且必须平衡有效的G.T修复和避免未损坏的DNA的需求，这可能会限制其活性。鉴于生物学需要维持CPG的避免突变和转录调节的完整性，重要的是要详细了解TDG和MBD4如何识别和消除病变，为什么它们的活性对G.T失误（这可能会影响CPG突变性）以及其活性如何受到APE1的调节。为此，我们提出了生化，生物物理和结构方法的有力组合，以实现四个具体的目标：（i）我们将使用瞬态动力学和平衡结合方法来确定病变识别，病变切除和产品释放的病变和MBD4的速度和g.t lie的速度slosing g。尿嘧啶，例如5FU。 （ii）我们将确定与含有不可裂解的底物类似物的DNA结合的TDG（催化结构域）的晶体结构，从而揭示了促进G.T特异性的相互作用。为了了解TDG如何询问但不对CPG站点作用，我们将尝试解决与CpG DNA结合的TDG的结构。 （iii）我们将使用具有稳定的TDG-AP-DNA复合物的瞬时动力学和NMR方法来揭示APE1如何调节TDG活性（增强其周转率）。我们还将检验以下假设：及时释放产品和有效修复G.T病变所必需的TDG的Sumoylation。 （iv）我们将使用NMR来确定TDG的内在无序的N末端区域如何实现有效的G.T修复，以及无序的C末端区域如何与Sumo蛋白形成非共价相互作用，这对于SUMO调节TDG活性和TDG的（TDG）与Sumo-My-Modifiend蛋白（I.E.E.，P731，PML）。了解本质上无序的蛋白质区域的功能是结构生物学的主要挑战，我们的研究将有助于这一新兴领域。这些研究的成功完成将提高我们对人类识别和修复诱变G.T病变的理解，为什么CpG站点是突变的热点，以及TDG如何介导5FU的细胞毒性（一种广泛使用的抗癌药物），对TDG和MBD4在癌症中的作用含义，伴有癌症和MBD4在癌症中的作用。