Elucidating Mechanisms of Recognition and Excision of Damaged Bases by NEIL glycosylases

阐明 NEIL 糖基化酶识别和切除受损碱基的机制

• 批准号: 10462636
• 负责人: SHEILA Sue DAVID
• 金额: $ 28.61万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462636
• 关键词: Address; Aging; Amino Acids; Arginine; Base Excision Repairs; Binding; Biological Assay; Cells; Cellular Assay; Chemistry; Codon Nucleotides; Crystallization; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Repair Gene; DNA Sequence Alteration; DNA lesion; Defect; Deoxyribose; Detection; Enzymatic Biochemistry; Enzymes; Excision; Flow Cytometry; Fluorescence Microscopy; Functional disorder; G-Quartets; Gene Expression; Genes; Genetic Transcription; Genome; Glycosides; Hydantoins; In Vitro; Kinetics; Laboratories; Lesion; Link; Lysine; Maintenance; Malignant Neoplasms; Mammalian Cell; Mediating; Molecular; Monitor; NEIL3 gene; Nucleic Acids; Nucleotides; Oncogenes; Oxidative Stress; Oxides; Plasmids; Play; Positioning Attribute; Process; Promoter Regions; Property; Protein Isoforms; Pyrimidines; RNA Editing; Reaction; Reporter; Roentgen Rays; Role; Scanning; Signal Transduction; Site; Structure; Substrate Specificity; Transact; base; biophysical techniques; experimental study; genome integrity; guanidinohydantoin; malignant neurologic neoplasms; nervous system disorder; oxidation; prevent; promoter; recruit; repaired; response; single molecule; thymine glycol; trend

Oxidative stress erodes the integrity of DNA by modifying DNA bases and has been linked to cancer, neurological disorders and aging. The NEIL glycosylases initiate base excision repair of oxidized base lesions by catalyzing the cleavage of the N-glycosidic linkage to the 2’-deoxyribose and are capable of removing a wide array of modified DNA bases. The NEIL glycosylases are unique in acting in a variety of different contexts beyond duplex DNA, such as ssDNA, and G-quadruplexes, that has suggested that these enzymes play central roles in repair, replication and transcription. We have shown that hydantoin lesions, that are formed formed under conditions of high oxidative stress, and in G-rich sequences such as G-quadruplexes, are the best substrates for the NEIL glycosylases. Our laboratory was also the first to provide a direct link between RNA editing and DNA repair by showing lesion processing by NEIL1 is modulated by an RNA editing reaction that changes the codon for amino acid position 242 in the lesion recognition loop of the enzyme, switching the residue from the genomically encoded lysine to an arginine. This change alters NEIL1 glycosylase rate constants in a lesion and DNA context dependent manner. Lesion identity and context also influences the extent of NEIL1 base excision, and this lesion binding property suggest roles in regulating replication and transcription. We have also uncovered unique differences between the NEIL1 and NEIL3 in the removal of hydantoin lesions from different G-quadruplex sequences. The presence of different G-quadruplex sequences in gene promoters, curiously in the DNA repair enzymes themselves, suggests that the observed differences in extents of lesion excision, and the presence of non-productive binding, may alter transcription in response to oxidative stress. These observations further implicate NEIL enzymes in processes beyond classic BER, and further underscore the tight interdependence of nucleic acid transactions. This project will entail using a multi- faceted approach involving enzymology, nucleic acid chemistry, biophysical methods and cellular assays to probe the lesion and context dependent properties of NEIL1, 2 and 3 to make direct connections between molecular defects in particular aspects of damage recognition and base excision on preventing DNA mutations and altering gene transcription.

氧化应激通过修饰DNA碱基侵蚀DNA的完整性，并与癌症有关， 神经系统疾病和衰老。尼尔糖基酶启动了氧化物基本分泌的基本惊喜修复 通过催化N-糖苷键的裂解至2'-脱氧核糖，并能够去除A 广泛的修饰DNA碱基。尼尔糖基酶在各种不同的作用方面都是独特的 双链DNA（例如ssDNA和G-四链体）以外的上下文表明这些酶 在维修，复制和转录中扮演核心角色。我们已经证明了Hydantoin病变，那就是 在高氧化应激条件下形成的形成，在诸如g四链体等富含G的序列中形成的是 尼尔糖基酶的最佳底物。我们的实验室也是第一个提供直接联系的人 RNA编辑和DNA修复通过显示Neil1的病变处理通过RNA编辑反应调节 这会改变酶病变识别环中氨基酸位置的密码子242，从而切换 从基因组编码的赖氨酸到精氨酸的残基。这种变化改变了neil1糖基酶速率 病变和DNA上下文依赖性方式的常数。病变身份和背景也影响 Neil1基本的范围令人惊讶，而这种病变结合特性表明在调节复制和 转录。我们还发现了Neil1和Neil3之间的独特差异 来自不同G-四链体序列的氢卫生病变。存在不同的G四链体序列 在基因启动子中，在DNA修复酶本身中奇怪地表明，观察到的差异 病变惊喜的范围以及存在非生产性结合的存在可能会改变转录 氧化应激。这些观察结果进一步暗示了尼尔酶在经典BER之外的过程中，以及 进一步强调了核酸交易的紧密相互依赖。该项目将需要使用多个 涉及酶学，核酸化学，生物物理方法和细胞测定法 探测Neil1、2和3的病变和上下文依赖性特性，以在之间建立直接连接 损害识别的特定方面的分子缺陷和防止DNA突变的基本惊喜 并改变基因转录。