UNDERSTANDING HOMOLOGY IN GENETIC RECOMBINATION

了解基因重组中的同源性

• 批准号: 6019486
• 负责人: SCOTT F SINGLETON
• 金额: $ 18.77万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019486
• 关键词: affinity labeling; chemical stability; enzyme substrate complex; fluorescent dye /probe; genetic recombination; nucleoside analog; protein binding; recombinase; site directed mutagenesis; stop flow technique

DESCRIPTION: Homologous genetic recombination is a fundamental biological process that is well appreciated at a formal genetic level: it plays central roles in inheritance and the proper segregation of chromosomes, but is also important in DNA repair, replication, regulation, and differentiation. In addition, the specific introduction of foreign DNA into eukaryotic chromosomes a basic step in the developing are of gene therapy, is critically dependent on genetic recombination machinery. The RecA protein of E. coli has served as a recombinase prototype, and has been the most widely investigated member of this universal class of proteins. Although RecA has been the subject of biological research for 30 years, the molecular mechanisms of the genetic exchange events remain unclear. The long-term goal of the research proposed herein is to understand in molecular terms the structural and dynamic mechanisms of genetic recombination, the processes by which the genetic information is reorganized by the physical rearrangement of elemental pieces of the genetic material. During the project period to be supported by an NIH Research Grant, the research is directed towards significant and novel contributions to the molecular understanding of RecA-mediated recombination by characterizing the forces of molecular recognition that control recombination events. Bio-organic and biophysical approaches will be used to examine the equilibrium and kinetics of the interactions that are important for recombination. In order to dissect the molecular steps involved in recombination, 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 agents and site- directed mutagenesis will elucidate the molecular mechanisms by which RecA binds DNA an allows sequence homology to be recognized. Ultimately, we hope to exploit this understanding in vivo through the control and directed manipulation of RecAs function.

描述： 同源基因重组是一种基本的生物学 在正式的遗传水平上受到广泛赞赏的过程：它起着 在遗传和染色体正确分离中发挥核心作用， 但在 DNA 修复、复制、调节和 差异化。 另外，具体引入外源DNA 进入真核染色体是基因发育的基本步骤 治疗，很大程度上依赖于基因重组机制。 这 大肠杆菌的 RecA 蛋白已作为重组酶原型，并具有 是这个普遍类别中研究最广泛的成员 蛋白质。尽管 RecA 30 年来一直是生物学研究的主题 多年来，遗传交换事件的分子机制仍然存在 不清楚。 本文提出的研究的长期目标是 从分子角度理解其结构和动力学机制 基因重组，遗传信息重组的过程 通过对元素的物理重新排列进行重组 遗传物质。 项目期间将得到 NIH 的支持 研究补助金，该研究针对重要且新颖的 对 RecA 介导的分子理解的贡献 通过表征分子识别力进行重组 控制重组事件。 生物有机和生物物理方法将 用于检查相互作用的平衡和动力学 对于重组很重要。 为了剖析分子步骤 参与重组，我们最初的努力将集中在关键的早期 事件：DNA 结合和底物 DNA 之间同源性的搜索。 因为重组是一个主动的过程，涉及难以捉摸的中介 蛋白质-DNA复合物，该研究将重点关注 使用停流剂和位点的前稳态动力学方法 定向诱变将阐明 RecA 的分子机制 结合 DNA 并允许识别序列同源性。最终，我们 希望通过控制和利用体内的这种理解 RecAs 函数的定向操作。