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很大比例的突变是CpG位点的CAET转变，主要由5-甲基胞嘧啶（m5C）脱胺导致G.T错配而产生。CpG位点的胞嘧啶甲基化是转录沉默的标志，这是许多细胞过程的中心，也是胚胎发生所必需的。因此，维持CpG的完整性对于避免突变和适当的转录调控是重要的。胸腺嘧啶DNA糖基酶（TDG）和甲基结合结构域IV （MBD4）是识别G.T病变的两种DNA糖基酶。这些糖基酶启动碱基切除修复（BER）途径，将目标核苷酸（dT）翻转到它们的活性位点，并切割碱基糖（n -糖基）键，产生碱基（AP）位点。AP内切酶（APE1）和下游BER酶继续修复。TDG和MBD4面临着从错配对中去除正常碱基的艰巨任务，必须平衡有效的G.T修复需求和避免未受损的DNA，这可能会限制它们的活性。考虑到维持CpG完整性以避免突变和转录调控的生物学需要，详细了解TDG和MBD4如何识别和清除病变，为什么它们的活性对于G.T错配（可能影响CpG的易变性）很慢，以及它们的活性如何被APE1调节是很重要的。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TDG和MBD4病变识别、病变切除和产品释放的参数，揭示为什么它们的转换在G.T病变中缓慢，而在5-卤化尿嘧啶（如5FU）的切除中快速。（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如何询问而不作用于CpG位点，我们将尝试解决与CpG DNA结合的TDG的结构。（iii）我们将使用瞬态动力学和稳定的TDG- ap - dna复合物的NMR方法来揭示APE1如何调节TDG活性（增强其周转率）。我们还将验证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内在无序的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SUMO调节TDG活性和TDG与SUMO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促进我们对人类突变性G.T病变如何被识别和修复，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TDG和MBD4在癌症、遗传性疾病和5FU化疗中的作用具有指导意义。

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.