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 基因组完整性并介导表观遗传调控，以及 BER 缺陷如何影响人类健康。一个 主要重点是发现启动 BER 的 DNA 糖基化酶如何发现并切除受损或修饰的 5-甲基胞嘧啶（mC）的形式。 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 植物和动物的调节以及古细菌和细菌的限制修饰。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的完整性并导致包括癌症在内的人类疾病。反击这一点 威胁是三种不同类型的糖基化酶，它们可以从 G/T 错配中切除 T；哺乳动物中的 TDG 和 MBD4 和古细菌和细菌中的 MIG。虽然大多数糖基化酶会切除 DNA 外源碱基（例如尿嘧啶） 这些酶面临着去除 mC 脱氨产生的胸腺嘧啶碱基的艰巨任务，但不是那些在 A:T 对或聚合酶产生的 G/T 错配的广阔背景。因为糖基化酶作用于 未受损的 DNA 具有诱变性，这些 G/T 糖基化酶的特异性至关重要，但目前尚不清楚。这 当前的范式认为特异性涉及对不匹配的鸟嘌呤的识别。我们将严格测试 该模型并研究其他潜在的特异性因素，以确定 G/T 糖基化酶的机制 特异性。我们的研究将揭示 TDG 和 MBD4 的特征，这些特征可能是 mC 修复效率低下的原因 脱氨基作用是癌症和遗传病中点突变的潜在原因。 BER 也起作用 通过主动 DNA 去甲基化来“擦除”mC，从而参与表观遗传调控。已建立的途径 在脊椎动物中，mC 被 TET 酶氧化，产生三种氧-mC 产物（hmC、fC、caC）， 通过 TDG 切除 fC 或 caC，以及随后的 BER 以产生未经修改的 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，以及 它如何被招募到 DNA 去甲基化位点。我们还对翻译后修饰如何 调节 BER，我们当前的重点是确定 SUMO 修改如何调节 TDG。 TDG 是 在单个位点进行苏酰化，并且它具有结合 SUMO 结构域的 SUMO 相互作用基序 (SIM)，其中包括 分子内相扑。虽然 TDG 被认为是了解 sumoylation 如何调节的模型 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 如何调节这些影响。我们还将定义 SUMO 共轭和机制 解共轭并了解 SIM 如何调节这些过程。体外结合-解结合 系统将用于测试 TDG 的苏酰化需要调节其产品释放的范例 并确保忠实完成TDG发起的BER。这些研究的结果将揭示 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。