Understanding Homology in Genetic Recombination

了解基因重组中的同源性

• 批准号: 6792767
• 负责人: SCOTT F SINGLETON
• 金额: $ 29.78万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-10-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6792767
• 关键词: DNA; DNA repair; Escherichia coli; affinity labeling; binding sites; chemical stability; electron spin resonance spectroscopy; enzyme substrate complex; fluorescence resonance energy transfer; fluorescent dye /probe; gene targeting; genetic recombination; molecular shape; nucleic acid structure; nucleoside analog; protein binding; recombinase; restriction fragment length polymorphism; site directed mutagenesis; stop flow technique

DESCRIPTION (provided by applicant): In living organisms, there is a dynamic balance between the need to preserve genomic information and the need to generate genetic diversity. The repair of damaged DNA is essential to the maintenance of heritable genetic information, while the variation of that information drives evolutionary adaptation. The E. coli RecA protein mediates events in both pathways, and because its functions are conserved from bacteriophage to humans, its study has provided a paradigm for understanding these essential biological processes. The molecular mechanisms underlying the complex process of DNA strand pairing and exchange are not yet understood. In particular, there is still substantial uncertainty with regard to the role of the sequence-dependent DNA-DNA interactions that are the hallmark of homologous recombination. In order to delimit possible models for recombinational activities, we propose to investigate the mechanism of the DNA strand exchange reaction mediated by the RecA protein. During the project period to be supported by an NIH Research Grant, the research is directed towards the characterization of site-specific interactions among the reaction constituents in order to reach a molecular understanding of recombination and recombinational repair by the construction and elaboration of three-dimensional models of the intermediates in the reactions. Bioorganic and biophysical approaches will be used to examine the equilibrium and kinetics of the interactions that are important for recombination. In order to tease apart the molecular events, our initial efforts will focus on key early events. DNA binding and the search for homology between substrate DNAs. Because recombination is an active process involving elusive intermediary protein-DNA complexes, the research will focus a major emphasis on a presteady-state kinetic approach using stopped-flow spectrofluoriometry. In combination with affinity labeling, time-resolved FRET structural characterization, and EPR dynamic characterization, transient kinetics agents will elucidate the molecular mechanisms by which RecA binds DNA and allows sequence homology to be recognized. Ultimately, we hope to exploit this understanding in vivo through the control and directed manipulation of RecA's function.

描述（由申请人提供）：在生物体中，保存基因组信息的需要和产生遗传多样性的需要之间存在动态平衡。 受损 DNA 的修复对于维持可遗传的遗传信息至关重要，而该信息的变异则驱动进化适应。 大肠杆菌 RecA 蛋白介导这两种途径中的事件，并且由于其功能从噬菌体到人类都是保守的，因此其研究为理解这些重要的生物过程提供了范例。 DNA 链配对和交换的复杂过程背后的分子机制尚不清楚。 特别是，作为同源重组标志的序列依赖性 DNA-DNA 相互作用的作用仍然存在很大的不确定性。 为了界定重组活动的可能模型，我们建议研究 RecA 蛋白介导的 DNA 链交换反应的机制。 在美国国立卫生研究院研究基金支持的项目期间，该研究旨在表征反应成分之间的位点特异性相互作用，以便通过构建和阐述三维结构来达到对重组和重组修复的分子理解。反应中的中间体模型。 生物有机和生物物理方法将用于检查对于重组很重要的相互作用的平衡和动力学。 为了梳理分子事件，我们最初的努力将集中于关键的早期事件。 DNA 结合和底物 DNA 之间同源性的搜索。 由于重组是一个涉及难以捉摸的中间蛋白质-DNA 复合物的主动过程，因此该研究将重点关注使用停流荧光光谱法的前稳态动力学方法。 结合亲和标记、时间分辨 FRET 结构表征和 EPR 动态表征，瞬态动力学试剂将阐明 RecA 结合 DNA 并允许识别序列同源性的分子机制。 最终，我们希望通过控制和定向操纵 RecA 功能来在体内利用这种理解。