Understanding Homology in Genetic Recombination

了解基因重组中的同源性

基本信息

批准号：
8289578
负责人：
SCOTT F SINGLETON
金额：
$ 31.29万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-10-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8289578
关键词：
ATP Hydrolysis Achievement Address Adjuvant Affect Affinity Amino Acid Sequence Anti-Bacterial Agents Antibiotic Resistance Antibiotics Attenuated Bacteria Bacterial Drug Resistance Binding Biochemical Biological Assay Biological Process Cells Cellular Assay Collection Communicable Diseases Competence Complex DNA DNA Binding Data Development Dissection Enzyme Kinetics Escherichia coli Evaluation F Factor Filament Funding Gene Mutation Gene Transfer Genes Genetic Recombination Goals In Vitro Individual Interdisciplinary Study Kinetics Knowledge Laboratories Legal patent Life Ligands Light Literature Mediating Methods Modeling Molecular Motor Mutagenesis Mutation Organism Pathogenicity Pathway interactions Peptide Sequence Determination Pharmacology Physiological Play Polymerase Gene Process Property Proteins Protocols documentation Public Health Reaction Rec A Recombinases Recombinants Research Resistance Resolution Role SOS Response Signal Transduction Site Son of Sevenless Proteins Stress Structure Structure-Activity Relationship Testing Therapeutic Index Thermodynamics Translating Variant Zidovudine analog antineoplastic antibiotics bactericide biodefense biothreat cell killing chemotherapy combat design homologous recombination in vitro activity in vivo inhibitor/antagonist insight intein interest novel nucleotide analog pathogen pathogenic bacteria programs public health relevance rapid technique response small molecule tool transmission process

项目摘要

DESCRIPTION (provided by applicant): Antibiotic resistance is an escalating problem for modern chemotherapy of bacterial infectious diseases, and, in combination with the deteriorating pipeline of new antibacterials, is creating a clear and urgent danger to public health and national biodefense. Although the mechanisms that facilitate the de novo development, clonal spread, and horizontal transfer of resistance factors are not fully understood, the rapid rate at which antibiotic-resistant bacteria arise is likely due to a combination of mutations introduced during SOS mutagenesis and gene transfer between organisms. Recently, the Escherichia coli RecA protein's activities in SOS induction and homologous recombination have revealed RecA as a crucial player in these phenomena. A combination of primary literature, patent data, and unpublished results demonstrate that RecA mediates a range of phenomena related to bacterial pathogenecity, particularly the development and transmission of antibiotic resistance genes. Although the high conservation of RecA among bacterial species compellingly suggests the possibility that RecA may play similar roles in species other than E. coli, many questions remain as to how the properties of individual variants are related to their specific biological functions. To delimit possible models for the RecA-mediated activities that occur in pathogenic bacteria, we propose three Specific Aims to exploit our recently developed methods for rapid, parallel purification and rigorous characterization of RecA proteins to elucidate the relationships between RecA structure, in vitro activities, and physiologic functions. Briefly, we will (1) systematically define and evaluate structure-function relationships among RecA proteins from 31 pathogenic bacteria using biochemical and cellular activity assays; (2) provide insight into the species-specific molecular mechanisms of RecA- DNA filament activation using directed mutagenesis and substrate analogs; and (3) demonstrate that RecA effectors can be delivered into living bacteria to produce physiological consequences. The successful realization of the Aims will provide (1) substantial and novel insights into the molecular mechanisms by which different RecA proteins from select bacterial pathogens carry out their biological functions; (2) a novel microbiological toolbox that will be central to teasing apart the various roles of RecA in pathogenicity; and (3) novel methods for the delivery of small-molecule RecA effectors into bacterial pathogens. PUBLIC HEALTH RELEVANCE: The bacterial RecA protein is an emerging target for adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index. By combining the use of enzyme kinetics and cellular assays with the synthesis of small-molecule ligands to probe structure-function relationships among RecA proteins from select pathogenic bacteria, a greater understanding of these proteins' roles in various aspects of bacterial pathogenicity - including the de novo development and transmission of antibiotic resistance genes - will be obtained. This new knowledge will increase the efficiency of the discovery of small-molecule effectors that address the urgent unmet need to overcome antibiotic resistance in biothreat and other pathogenic bacteria.

描述（由申请人提供）：抗生素耐药性是细菌传染病现代化疗面临的一个日益严重的问题，并且与新型抗菌药物的不断恶化相结合，正在对公共卫生和国家生物防御造成明显而紧迫的危险。尽管促进耐药因子从头发育、克隆传播和水平转移的机制尚不完全清楚，但抗生素耐药性细菌的快速出现可能是由于 SOS 诱变和基因转移过程中引入的突变组合所致。有机体。最近，大肠杆菌 RecA 蛋白在 SOS 诱导和同源重组中的活性表明 RecA 在这些现象中发挥着关键作用。原始文献、专利数据和未发表结果的结合表明，RecA 介导一系列与细菌致病性相关的现象，特别是抗生素抗性基因的发展和传播。尽管RecA在细菌物种中的高度保守性令人信服地表明RecA可能在大肠杆菌以外的物种中发挥相似的作用，但关于单个变体的特性如何与其特定的生物学功能相关，仍然存在许多问题。为了界定病原菌中 RecA 介导的活性的可能模型，我们提出了三个具体目标，以利用我们最近开发的方法对 RecA 蛋白进行快速、平行纯化和严格表征，以阐明 RecA 结构、体外活性、和生理功能。简而言之，我们将 (1) 使用生化和细胞活性测定系统地定义和评估来自 31 种病原菌的 RecA 蛋白之间的结构-功能关系； (2) 使用定向诱变和底物类似物深入了解 RecA-DNA 丝激活的物种特异性分子机制； (3) 证明 RecA 效应器可以被传递到活细菌中以产生生理后果。这些目标的成功实现将提供（1）对来自选定细菌病原体的不同 RecA 蛋白发挥其生物学功能的分子机制的实质性和新颖的见解； (2) 一个新的微生物工具箱，它将成为梳理 RecA 在致病性中的各种作用的核心； (3) 将小分子 RecA 效应物递送到细菌病原体中的新方法。公共卫生相关性：细菌 RecA 蛋白是抗生素化疗佐剂的新兴靶标，可调节抗生素耐药基因的发展和传播并增加抗生素治疗指数。通过将酶动力学和细胞测定与小分子配体的合成相结合来探测来自选定病原菌的 RecA 蛋白之间的结构-功能关系，可以更好地了解这些蛋白在细菌致病性各个方面的作用 - 包括 de抗生素抗性基因的新发展和传播 - 将获得。这一新知识将提高小分子效应物发现的效率，解决克服生物威胁和其他病原菌抗生素耐药性的迫切未满足的需求。