Mechanisms of BER in Genomic Integrity and Epigenetic Regulation

BER 在基因组完整性和表观遗传调控中的机制

• 批准号: 10726878
• 负责人: Alex C Drohat
• 金额: $ 4.48万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10726878
• 关键词: Aberrant DNA Methylation; Aging; Animals; Archaea; Bacteria; Base Excision Repairs; Binding; Cytosine; DNA; DNA Methylation; DNA Repair Enzymes; DNA glycosylase; Deamination; Disease; Ensure; Enzymes; Epigenetic Process; Excision; Face; Gene Expression; Genetic Diseases; Genomics; Goals; Guanine; Health; Human; In Vitro; Learning; Malignant Neoplasms; Mammals; Mediating; Methylation; Modeling; Modification; Mutation; Nature; Pathway interactions; Plants; Point Mutation; Polymerase; Post-Translational Protein Processing; Process; Research; Site; Specificity; Sumoylation Pathway; System; Testing; Thymine; Uracil; Vertebrates; base; demethylation; enzyme activity; epigenetic regulation; genome integrity; human DNA; human disease; interest; novel therapeutic intervention; oxidation; recruit; repair enzyme; repaired

An overarching goal of our research is to understand how the base excision repair (BER) pathway maintains genomic integrity and mediates epigenetic regulation, and how deficiencies in BER impact human health. A major focus is to discover how DNA glycosylases, which initiate BER, find and excise damaged or modified forms of 5-methylcytosine (mC). The most abundant modified DNA base in nature, mC is critical for epigenetic regulation in plants and animals and for restriction modification in archaea and bacteria. However, cytosine methylation also poses a danger because mC deaminates to T, generating G/T mispairs and C to T mutations that threaten genomic and epigenetic integrity and causes human diseases including cancer. Countering this threat are three different types of glycosylases that excise T from G/T mispairs; TDG and MBD4 in mammals and MIG in archaea and bacteria. While most glycosylases excise bases that are foreign to DNA (e.g., uracil) these enzymes face the daunting task of removing thymine bases arising by mC deamination but not those in the vast background of A:T pairs or in polymerase-generated G/T mispairs. Because glycosylase action on undamaged DNA is mutagenic, the specificity of these G/T glycosylases is critical, yet it is poorly defined. The current paradigm holds that specificity involves recognition of the mismatched guanine. We will rigorously test this model and investigate other potential specificity factors, to define the mechanism of G/T glycosylase specificity. Our studies will reveal features of TDG and MBD4 that may account for inefficient repair of mC deamination, a potential cause of point mutations implicated in cancer and genetic disease. BER also functions in epigenetic regulation by serving to “erase” mC through active DNA demethylation. An established pathway in vertebrates involves oxidation of mC by a TET enzyme to give three oxy-mC products (hmC, fC, caC), excision of fC or caC by TDG, and subsequent BER to yield unmodified C. Our studies will address major gaps in the understanding of this essential pathway, by defining how TDG recognizes and removes fC and caC and how it is recruited to sites of DNA demethylation. We are also interested in how post-translational modifications regulate BER, and our current focus is on determining how TDG is regulated by SUMO modification. TDG is sumoylated at a single site, and it has a SUMO-interacting motif (SIM) that binds SUMO domains, including an intramolecular SUMO. While TDG is considered a model for understanding how sumoylation can regulate enzyme activity, many fundamental questions remain. Our studies will reveal how sumoylation alters TDG activity and how the SIM mediates these effects. We will also define mechanisms of SUMO conjugation and deconjugation and learn how the SIM modulates these processes. An in vitro conjugation-deconjugation system will be used to test the paradigm that sumoylation of TDG is required to regulate its product release and ensure faithful completion of TDG-initiated BER. Results of these studies will inform how BER deficiencies impact human health and could suggest new therapeutic approaches for treating diseases including cancer.

我们研究的首要目标是了解碱基切除修复（BER）途径如何维持 基因组完整性和介导表观遗传调控，以及BER缺陷如何影响人类健康。一 主要的焦点是发现启动BER的DNA糖基化酶如何发现和切除受损或修饰的DNA， 5-甲基胞嘧啶（mC）。mC是自然界中最丰富的修饰DNA碱基，对于表观遗传至关重要。 在植物和动物中的调节以及在古细菌和细菌中的限制性修饰。然而，胞嘧啶 甲基化也会带来危险，因为mC脱氨基为T，产生G/T错配和C到T突变 威胁基因组和表观遗传完整性并导致包括癌症在内的人类疾病。打击这一 威胁是三种不同类型的糖基化酶，它们从G/T错配对中切除T;哺乳动物中的TDG和MBD4 以及古细菌和细菌中的微生物。虽然大多数糖基化酶切除DNA外源的碱基（例如，尿嘧啶） 这些酶面临着艰巨的任务，即除去由mC脱氨基作用产生的胸腺嘧啶碱基，而不是那些 A：T配对或聚合酶产生的G/T错配的巨大背景。因为糖基化酶作用于 未受损的DNA是致突变的，这些G/T糖基化酶的特异性是至关重要的，但它的定义很差。的 目前的范例认为特异性包括识别错配的鸟嘌呤。我们将严格测试 本研究通过建立该模型，并探讨其他可能的特异性因子，以明确G/T糖基化酶的作用机制 的特异性我们的研究将揭示TDG和MBD 4的特征，这些特征可能解释了mC修复效率低下的原因。 脱氨基，一个潜在的点突变的原因牵连在癌症和遗传疾病。BER也起作用 在表观遗传调控中，通过主动DNA去甲基化来"清除" mC。一个既定的途径 在脊椎动物中，涉及通过泰特酶氧化mC以产生三种氧-mC产物（hmC、fC、caC）， 通过TDG切除fC或caC，随后通过BER产生未修饰的C。我们的研究将解决主要差距 通过定义TDG如何识别和去除fC和caC， 它是如何被募集到DNA去甲基化位点的。我们也对翻译后修饰 调节BER，我们目前的重点是确定如何通过SUMO修饰调节TDG。TDG是 在单个位点被SUMO化，并且它具有结合SUMO结构域的SUMO相互作用基序（SIM），包括SUMO结构域。 分子内相扑虽然TDG被认为是理解类小泛素化如何调节 酶活性，许多基本问题仍然存在。我们的研究将揭示sumoylation如何改变TDG 活动以及SIM如何介导这些效应。我们还将定义SUMO结合的机制， 了解SIM如何调节这些过程。体外结合-去结合 系统将用于测试TDG需要SUMO化来调节其产物释放的范式 并确保TDG发起的BER的忠实完成。这些研究的结果将告知BER缺陷如何 影响人类健康，并可能为治疗包括癌症在内的疾病提出新的治疗方法。