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: 10605583
• 负责人: Alex C Drohat
• 金额: $ 5.98万
• 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/10605583
• 关键词: Aberrant DNA Methylation; Address; 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; cancer genetics; demethylation; enzyme activity; epigenetic regulation; genome integrity; human DNA; human disease; interest; novel therapeutic intervention; oxidation; recruit; repair enzyme; repaired; Aberrant DNA Methylation; Address; 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; cancer genetics; 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.

我们研究的总体目标是了解基本惊喜维修（BER）途径如何保持 基因组完整性并介导表观遗传调节，以及BER的缺陷如何影响人类健康。一个 主要重点是发现启动BER，查找和消除受损或修饰的DNA糖基酶如何 5-甲基胞嘧啶（MC）的形式。自然界中最丰富的修饰DNA碱基，MC对于表观遗传至关重要 植物和动物的调节，以及用于古细菌和细菌的限制修饰。但是，胞嘧啶 甲基化也构成了危险 这威胁到基因组和表观遗传完整性，并引起包括癌症在内的人类疾病。反对这个 威胁是三种不同类型的糖基酶，这些糖基酶经历了g/t遗失的T；哺乳动物中的TDG和MBD4 和古细菌和细菌中的MIG。虽然大多数糖基酶消除了DNA属于DNA的碱（例如，uracil） 这些酶面临着去除MC死亡产生的胸腺素基础的艰巨任务，而不是 a：t对或聚合酶生成的g/t的巨大背景。因为糖基酶对 未损坏的DNA是诱变的，这些G/T糖基化酶的特异性至关重要，但定义很差。这 当前的范式认为，特异性涉及对不匹配的鸟嘌呤的认识。我们将严格测试 该模型并研究其他潜在特异性因子，以定义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，这可能解释了MC的效率低下 脱氨酸，这是癌症和遗传疾病中实施的点突变的潜在原因。 BER也起作用 在表观遗传学调节中，通过活跃的DNA脱甲基化来“擦除” MC。已建立的途径 在脊椎动物中，涉及通过TET酶氧化MC以得到三种氧MC产物（HMC，FC，CAC）， 通过TDG切除FC或CAC，随后的BER产生未修饰的C。我们的研究将解决主要差距 在理解这一基本途径时，通过定义TDG如何识别和删除FC和CAC以及 如何将其招募到DNA脱甲基化的位置。我们也对翻译后的修改感兴趣 调节BER，我们目前的重点是确定TDG如何通过Sumo修饰调节。 TDG是 在一个位点上进行的sumoyped，它具有相扑基序（SIM），该基元（SIM）结合了Sumo域，包括一个 分子内相扑。虽然TDG被认为是了解如何调节sumoylation的模型 酶活性，仍然存在许多基本问题。我们的研究将揭示Sumoylation如何改变TDG 活动以及SIM如何介导这些效果。我们还将定义相扑结合的机制和 解耦合并了解SIM调制这些过程如何调节。体外施工解释 系统将用于测试范例，即需要TDG的Sumoylation来调节其产品释放 并确保忠实地完成TDG发起的BER。这些研究的结果将告知您如何缺陷 影响人类健康，并可能提出用于治疗包括癌症的疾病的新治疗方法。