SUBSTRATE SPECIFICITY OF THE BER OXIDATIVE DNA GLYCOSYLASES

BER 氧化 DNA 糖基化酶的底物特异性

基本信息

批准号：
7684717
负责人：
SUSAN S WALLACE
金额：
$ 34.7万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7684717
关键词：
Acids Algorithms Amino Acids Aspartate Base Excision Repairs Binding Biochemical Biological Biological Assay Cancer Patient Carcinogens Class Cleaved cell Complement Crystallization DNA DNA Binding Domain DNA Damage DNA glycosylase Deoxyribose Elements End Point Enzymes Escherichia coli Event Excision Repair Exhibits Family Family member Fluorescence Free Radicals Glycine Goals Helix (Snails)Human Hydrolysis Hydroxyl Radical Ionizing radiation Kinetics Knowledge Left Lesion Lyase Measurement Micrococcus luteus Modality Molecular Numbers Object Attachment Oligonucleotides Phylogenetic Analysis Play Process Proline Property Proteins Purines Pyrimidine Pyrimidine Dimers Pyrimidines Radiation Induced DNA Damage Range Reactive Oxygen Species Reader Resolution Role Site Specificity Standards of Weights and Measures Structure Substrate Specificity System Testing UV endonuclease Variant Water Work Zinc Fingers base carcinogenesis cytotoxicity ear helix endonuclease endonuclease III interest member paralogous gene phosphodiester purine repaired scaffold sound three dimensional structure

项目摘要

The long-term goal of this project is to understand the molecular mechanisms of substrate specificity in two families of enzymes that play a critical role in the repair of ionizing radiation damage: the HhH-GPD Nth Superfamily and the Fpg Family. The DNA damages recognized by these oxidative DNA glycosylases have been implicated in the initiation of carcinogenesis. The fundamental hypothesis underlying the proposed work is that the structural framework provided by any glycosylase superfamily or thmily member can support a wide variety of specificities for the excised base after substitution of a small number of residues. The specific aims are to determine the substrate specificities of a comprehensive set of members of the Nth Superfamily and Fpg Family of DNA glycosylases selected by phylogenetic/stmctural algorithms developed in Core A with the goal of identifying particular amino acid residues in the substrate binding pocket associated with recognition of specific substrates. Subsets of naturally occurring DNA glycosylases that have similar sequences but divergent substrate specificities, such as EcNth and Micrococcus luteus UV endonuclease (M1Uve) of the Nth SuperFamily as well as naturally occurring Fpg and Nei DNA glycosylases witl be examined. Specific amino acid residues in the substrate binding pockets of particular Nth Superfamity and Fpg Family members, targeted by Core A as involved in substrate recognition, will be altered and substrate binding properties examined. Standard steady state kinetic measurements will be determined with a set of oligonucleotide substrates that overlap those of all Nth Superfamily and Fpg Family members using a high throughput fluorescence plate reader in the Expression, Characterization and Crystallization Core. These biochemical studies wil] complement the structural studies in Project 2 and help target particular gtycosylases for crystallization. The combined resuks of Core A and Projects 1 and 2 should provide a sound basis for a better understanding of substrate specificity in these classes of DNA glycosylases.

该项目的长期目标是了解两个酶家族中底物特异性的分子机制，这些酶在修复电离辐射损伤中起着至关重要的作用：HHH-GPD NTH超级家族和FPG家族。这些氧化DNA糖基酶识别的DNA损害与癌发生有关。拟议工作的基本假设是，在替换少量残基后，任何糖基酶超家族或Thmily成员提供的结构框架可以为切除的基础提供多种特异性。具体目的是确定NTH超家族和FPG家族的一组综合成员的底物特异性和由核心A开发的系统发育/稳定算法选择的DNA糖基酶，其目的是鉴定与识别特定底物识别的底物结合中的特定氨基酸残基。具有相似序列但具有不同底物特异性的天然DNA糖基化酶的子集，例如Nth超家族的ECNTH和MICROCOCUS LUTEUS UV紫外核酸内核酸酶（M1UVE）受检查。特定nth的底物结合袋中的特定氨基酸残基由核心A涉及底物识别的核心A和FPG家族成员将改变并检查底物结合特性。标准的稳态动力学测量将用一组寡核苷酸底物确定，这些寡核苷酸底物使用高吞吐量荧光板读取器在表达，表征和结晶核心中重叠所有第N个超家族和FPG家族成员。这些生化研究将补充项目2中的结构研究，并有助于靶向特定的gtycosylases以进行结晶。核心A和项目1和2的组合架应为在这些类别的DNA糖基酶中更好地理解底物特异性提供合理的基础。