Structure and Mechanism of CpG specific DNA glycosylases

 描述(由申请人提供)：我们的长期目标之一是了解碱基切除修复(BER)如何维持表观遗传调控中的遗传完整性和功能。本项目旨在揭示启动BER的DNA糖基酶如何识别和去除DNA中脱胺或氧化形式的5-甲基胞嘧啶(MC)，以及它们的活性如何受到翻译后修饰的调节。MC是DNA中含量最丰富的修饰碱基，是真核生物基因沉默的表观遗传标记，在古生菌和细菌的限制性内切酶修饰系统中发挥作用。然而，胞嘧啶甲基化也对遗传和表观遗传的完整性构成严重威胁。MC到T的脱氨基产生G/T错配，在复制时，C到T的转变。通过这一过程，MC脱氨基导致了癌症和遗传病中很大一部分点突变，这突显了了解糖基酶如何识别和处理G/T错对的必要性。三种类型的糖基酶启动G/T错配修复：TDG(胸腺嘧啶DNA糖基酶)、MBD4(甲基结合结构域IV)和MIG(错配糖基酶)。虽然大多数糖基酶去除DNA外来的碱基(例如尿嘧啶)，但这些错配酶从罕见的G/T错配对中去除胸腺嘧啶，而不是从DNA中A：T对的巨大背景中去除胸腺嘧啶。由于糖基酶对未损伤的DNA的异常作用是诱变和细胞毒性的，这些酶的特异性是关键的，但它仍然知之甚少。目前的范例认为，错配特异性源于酶与错配的鸟嘌呤接触，但这一点仍未得到证实。使用实验和计算方法的协同组合，我们将测试这一模型并调查其他三个潜在的特异性因素，定义MBD4、MIG和TDG的G/T失配特异性的机制。最近的研究表明，Tet酶将MC氧化成5-羟甲基-C(HMC)、5-甲酰-C(FC)和5-羧基-C(CAC)，而Fc(或CAC)的TDG切除和后续的BER完成了Tet-TDG-BER的DNA去甲基化途径。TDG在表观遗传调控中的关键作用可能解释了TDG对胚胎发生的重要性。然而，最近发现的这种活性的分子基础还不清楚。新的高分辨率晶体结构将揭示TDG如何识别DNA中的FC和CAC。我们还将调查新发现的分子基础，即TDG相对于T(和其他尿嘧啶)在切割FC和CAC的碱基配对偏好上显示出巨大的差异，并调查TDG和BER处理相反链中近端FC或CAC碱基的能力，而不产生近端AP位点或双链断裂。TDG受到相扑修饰，它有一个相扑相互作用基序(SIM)，可以非共价结合相扑结构域。调节细胞中SUMO~TDG水平的机制还知之甚少。拟议的研究将揭示调节TDG的异肽酶，并确定它们的特异性和有效性。一种新的体外相扑修饰-去结合系统将被用来直接测试当前的TDG相扑甲基化调节产物释放和提高酶的周转的范式。

 DESCRIPTION (provided by applicant): One of our long-term goals is to understand how base excision repair (BER) maintains genetic integrity and functions in epigenetic regulation. This project aims to reveal how DNA glycosylases, which initiate BER, recognize and remove deaminated or oxidized forms of 5-methylcytosine (mC) from DNA, and how their activity is regulated by post-translational modification. As the most abundant modified base in DNA, mC serves as an epigenetic mark for gene silencing in eukaryotes and functions in the restriction modification systems of archaea and bacteria. However, cytosine methylation also poses a serious threat to genetic and epigenetic integrity. Deamination of mC to T generates G/T mispairs, and, upon replication, C to T transitions. Through this process mC deamination causes a large fraction of point mutations in cancer and genetic disease, which highlights the need to understand how glycosylases recognize and process G/T mispairs. Three types of glycosylases initiate repair of G/T mispairs: TDG (thymine DNA glycosylase), MBD4 (methyl binding domain IV), and MIG (mismatch glycosylase). While most glycosylases remove bases that are foreign to DNA (e.g., uracil), these mismatch enzymes remove thymine from rare G/T mispairs but not from the huge background of A:T pairs in DNA. Because aberrant glycosylase action on undamaged DNA is mutagenic and cytotoxic, the specificity of these enzymes is critical, but it remains poorly understood. The current paradigm holds that mismatch specificity derives from enzyme contacts with the mismatched guanine, but this remains unsubstantiated. Using a synergistic combination of experimental and computational methods, we will test this model and investigate three other potential specificity factors, defining the mechanism of G/T mismatch specificity for MBD4, MIG, and TDG. Recent studies show that TET enzymes oxidize mC, to 5-hydroxymethyl- C (hmC), 5-formyl-C (fC), and 5-carboxyl-C (caC), and that TDG excision of fC (or caC) and follow-on BER completes a TET-TDG-BER pathway for DNA demethylation. This key function in epigenetic regulation likely explains the essentiality of TDG for embryogenesis. However, the molecular basis of this recently discovered activity is poorly defined. New high-resolution crystal structures will reveal how TDG recognizes fC and caC in DNA. We will also investigate the molecular basis of new findings that TDG exhibits a vast difference in base- pairing preferences for excision of fC and caC relative to T (and other uracils), and investigate the capacity of TDG and BER to process proximal fC or caC bases in opposite strands without generating proximal AP sites or double-strand breaks. TDG is subject to SUMO modification and it has a SUMO-interacting motif (SIM) that binds non-covalently to SUMO domains. Mechanisms that regulate levels of SUMO~TDG in cells are poorly understood. The proposed studies will reveal isopeptidases that regulate TDG, and define their specificity and efficiency. A novel in vitro SUMO modification-deconjugation system will be used to directly test the current paradigm that sumoylation of TDG regulates product release and enhances enzymatic turnover.