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

基本信息

批准号：
10280321
负责人：
SHEILA Sue DAVID
金额：
$ 28.04万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/10280321
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

项目摘要

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.

氧化应激通过修改 DNA 碱基来破坏 DNA 的完整性，并与癌症有关，神经系统疾病和衰老。 NEIL 糖基化酶启动氧化碱基损伤的碱基切除修复。通过催化 N-糖苷键与 2'-脱氧核糖的断裂，能够去除 NEIL 糖基化酶具有多种不同的修饰 DNA 碱基，具有独特的作用。双链 DNA 之外的背景，例如 ssDNA 和 G-四链体，表明这些酶我们已经证明乙内酰脲损伤，即在修复、复制和转录中发挥核心作用。在高氧化应激条件下形成，并且在富含 G 的序列（例如 G 四链体）中，我们的实验室也是第一个提供 NEIL 糖基化酶之间直接联系的实验室。通过显示 NEIL1 的损伤处理受到 RNA 反应的调节来进行 RNA 编辑和 DNA 修复改变酶病变识别环中氨基酸位置 242 的密码子，从而切换残基从基因组编码的赖氨酸变为精氨酸，这种变化改变了 NEIL1 糖基化酶速率。病变和 DNA 背景相关的常数也影响着病变的身份和背景。 NEIL1 碱基切除的程度，这种损伤结合特性表明在调节复制和我们还发现了 NEIL1 和 NEIL3 在去除转录方面的独特差异。来自不同G-四链体序列的乙内酰脲损伤。在基因启动子中，奇怪的是在 DNA 修复酶本身中，表明观察到的差异病变切除的程度以及非生产性结合的存在可能会改变转录以响应这些观察结果进一步表明 NEIL 酶参与了经典 BER 之外的过程，并且进一步强调了核酸交易的紧密相互依赖性。涉及酶学、核酸化学、生物物理方法和细胞测定的多方面方法探测 NEIL1、2 和 3 的病变和上下文相关属性，以在 NEIL1、2 和 3 之间建立直接联系损伤识别和碱基切除特定方面的分子缺陷以防止 DNA 突变并改变基因转录。