Nucleic Acid Purification System

核酸纯化系统

• 批准号: 10797451
• 负责人: Alex C Drohat
• 金额: $ 11.39万
• 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/10797451
• 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; nucleic acid purification; 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.

我们研究的一个主要目标是了解碱基切除修复(BER)途径如何维持 基因组完整性和调节表观遗传调控，以及BER缺陷如何影响人类健康。一个 主要的焦点是发现启动BER的DNA糖基酶是如何发现和切除受损或修饰的 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，对表观遗传学至关重要 植物和动物的管制以及古生菌和细菌的限制性修改。然而，胞嘧啶 甲基化也是一种危险，因为MC脱氨基为T，产生G/T错配和C到T突变 这威胁到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酶，它们从G/T错配中剔除T；哺乳动物中的TDG和MBD4 古生菌和细菌中的MIG。虽然大多数糖基酶去除了DNA外来的碱基(如尿嘧啶) 这些酶面临着一项艰巨的任务，即去除由MC脱氨而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错配的广泛背景。因为糖基酶作用于 未损伤的DNA是突变的，这些G/T糖基酶的特异性是关键的，但它的定义很差。这个 目前的范式认为，特异性涉及识别不匹配的鸟嘌呤。我们将严格测试 并研究其他潜在的特异性因子，以明确G/T糖基酶的作用机制 专一性。我们的研究将揭示TDG和MBD4的特征，这可能是MC修复效率低下的原因 去氨基作用，这是癌症和遗传病中涉及的点突变的潜在原因。BER也有功能 在表观遗传调控中，通过活跃的DNA去甲基化来“擦除”MC。一条既定的道路 在脊椎动物中涉及通过Tet酶氧化MC以产生三种氧基-MC产物(HMC，FC，CAC)， 通过TDG切除FC或CAC，以及随后的误码率以产生未修改的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如何识别和移除FC和CAC以及 它是如何被招募到DNA去甲基化位点的。我们还感兴趣的是翻译后的修改如何 调节误码率，我们目前的重点是确定相扑修改如何调节TDG。TDG是 在单个位置相扑，它有一个相扑相互作用基序(SIM)，结合相扑结构域，包括一个 分子内相扑。而TDG被认为是一个理解相扑如何调控的模型 关于酶的活性，许多基本问题仍然存在。我们的研究将揭示相扑是如何改变tdg的。 活动以及SIM卡如何调节这些影响。我们还将定义相扑共轭机制和 了解SIM卡是如何调节这些过程的。一种体外结合去共轭的方法 系统将被用来测试TDG需要相加来调节其产品释放的范式 并确保忠实地完成TDG发起的BER。这些研究的结果将告诉我们误码率的不足 影响人类健康，并可能为治疗包括癌症在内的疾病提供新的治疗方法。