Structure and Mechanism of CpG specific DNA glycosylases

描述（由申请人提供）：遗传性疾病和癌症中的大部分突变是CpG位点的CAET转换，主要通过5-甲基胞嘧啶（m5 C）脱氨基产生G.T错配。在CpG位点的胞嘧啶甲基化是转录沉默的标志，其是许多细胞过程的中心并且是胚胎发生所必需的。因此，维持CpG完整性对于避免突变和适当的转录调控是重要的。两种DNA糖基化酶识别G.T病变，胸腺嘧啶DNA糖基化酶（TDG）和甲基结合结构域IV（MBD 4）。启动碱基切除修复（BER）途径，这些糖基化酶将靶核苷酸（dT）翻转到其活性位点，并切割碱基-糖（N-糖基）键，产生脱碱基（AP）位点。修复继续与AP核酸内切酶（APE 1）和下游BER酶。TDG和MBD 4面临着从错配对中移除正常碱基的艰巨任务，并且必须平衡有效G.T修复和避免未受损DNA的需求，这可能会限制它们的活性。鉴于生物学需要保持CpG完整性以避免突变和转录调控，重要的是要详细了解TDG和MBD 4如何识别和消除病变，为什么它们的活性对于G.T错配（可能影响CpG突变性）缓慢，以及它们的活性如何受APE 1调节。为此，我们提出了生物化学，生物物理学和结构方法的强大组合，以实现四个特定的目标：（i）我们将使用瞬态动力学和平衡结合方法来确定控制TDG和MBD 4的病变识别，病变切除和产物释放的参数，揭示为什么它们的营业额对于G.T病变缓慢，而对于切除5-卤代尿嘧啶如5 FU快速。(ii)我们将确定TDG（催化结构域）的晶体结构结合到DNA含有不可切割的底物类似物，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，我们将试图解决TDG与CpG DNA结合的结构。(iii)我们将使用瞬态动力学和NMR方法与稳定的TDG-AP-DNA复合物，以揭示APE 1如何调节TDG活性（增强其营业额）。我们还将检验TDG的SUMO化是及时释放产品和有效修复G.T病变所必需的假设。(iv)我们将使用NMR来确定TDG的固有无序的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TDG活性的SUMO调节和TDG与SUMO修饰的蛋白质的结合（即，p731，PML）。了解内在无序蛋白质区域的功能是结构生物学的一个重大挑战，我们的研究将有助于这一新兴领域。这些研究的成功完成将促进我们对人类如何识别和修复致突变G.T病变的理解，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5 FU的细胞毒性，以及TDG和MBD 4在癌症，遗传疾病和5 FU化疗中的作用。 公共卫生相关性：遗传性疾病和癌症中的大部分DNA突变可归因于5-甲基胞嘧啶（在CpG位点处）的脱氨基以产生G.T错配，尽管存在对这种损伤具有特异性的两种DNA修复酶TDG和MBD 4。这些研究将有助于我们进一步了解TDG和MBD 4如何识别和处理G.T病变，为什么它们的活性相对较慢，这可能会影响CpG突变性，以及它们的活性如何被下游修复酶APE 1增强。我们将确定TDG和MBD 4介导的5-氟尿嘧啶（5 FU）从DNA切除的动力学参数，进一步了解它们如何调节这种重要抗癌药物的细胞毒性。这些结果将增加我们对TDG和MBD 4在癌症、遗传性疾病和5 FU化疗中的作用的理解。

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. PUBLIC HEALTH RELEVANCE: A large percentage of the DNA mutations in genetic disease and cancer are attributable to deamination of 5-methylcytosine (at CpG sites) to give a G.T mispair, despite the presence of two DNA repair enzymes with specificity for such damage, TDG and MBD4. The proposed studies will advance our understanding of how TDG and MBD4 recognize and process G.T lesion, why their activity is relatively slow, which may impact CpG mutability, and how their activity is enhanced by APE1, a downstream repair enzyme. We will determine the kinetic parameters for TDG- and MBD4-mediated excision of 5-fluorouracil (5FU) from DNA, furthering our understanding of how they modulate the cytotoxicity of this important anticancer agent. The results will increase our understanding of the role of TDG and MBD4 in cancer, genetic disease, and 5FU chemotherapy.