STRUCTURAL BASIS OF ECO RI ENDONUCLEASE SPECIFICITY

ECO RI 核酸内切酶特异性的结构基础

• 批准号: 6226803
• 负责人: JOHN M ROSENBERG
• 金额: $ 24.86万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6226803
• 关键词: X ray crystallography; chemical kinetics; chemical structure function; computer simulation; crystallization; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; enzyme substrate complex; intermolecular interaction; mathematical model; molecular dynamics; nucleic acid chemical synthesis; nucleic acid structure; nucleotide analog; restriction endonucleases; site directed mutagenesis; structural biology; thermodynamics

The ultimate goals of this research are to elucidate molecular mechanisms of sequence-specific DNA-protein interactions as well as address broad issues of enzyme-substrate recognition and the molecular basis of specificity. This project proposes to continue structure-function analysis of Eco RI endonuclease, a restriction enzyme that recognizes the hexanucleotide GAATTC. This proposal combines X-ray crystallography and molecular dynamics calculations into a coordinated, structure-based effort to understand the relationship between structure and function in Eco RI endonuclease. The primary goals are: 1. To investigate Eco RI endonuclease-DNA complexes where the DNA contains base analog substitutions that perturb specific contacts between the protein and the DNA, e.g. by deleting or modifying individual moieties on the DNA. Both X-ray crystallography and molecular dynamics methods would be employed. 2. To similarly investigate Eco RI endonuclease-DNA complexes where the protein contains site-directed mutations that alter specific protein-DNA contacts. Both X-ray crystallography and molecular dynamics methods would be employed. 3. The investigate the structural basis for the known differences in binding energy caused by substitutions in the base-pairs immediately flanking the Eco RI site. Both X-ray crystallography and molecular dynamics methods would be employed. 4. To investigate the structural basis of sequence discrimination in Eco RI endonuclease by determining structures containing miscongnate or Eco RI sequences.

这项研究的最终目标是阐明序列特异性 DNA-蛋白质相互作用的分子机制，并解决酶-底物识别和特异性的分子基础的广泛问题。 该项目建议继续对 Eco RI 核酸内切酶进行结构功能分析，Eco RI 核酸内切酶是一种识别六核苷酸 GAATTC 的限制性内切酶。该提案将 X 射线晶体学和分子动力学计算结合到一个协调的、基于结构的工作中，以了解 Eco RI 核酸内切酶的结构和功能之间的关系。 主要目标是： 1. 研究 Eco RI 核酸内切酶-DNA 复合物，其中 DNA 含有扰乱蛋白质和 DNA 之间特定接触的碱基类似物取代，例如通过删除或修改 DNA 上的单个部分。 X射线晶体学和分子动力学方法都将被采用。 2. 类似地研究 Eco RI 核酸内切酶-DNA 复合物，其中蛋白质包含改变特定蛋白质-DNA 接触的定点突变。 X射线晶体学和分子动力学方法都将被采用。 3. 研究由紧邻 Eco RI 位点的碱基对的取代引起的已知结合能差异的结构基础。 X射线晶体学和分子动力学方法都将被采用。 4.通过确定含有错源或Eco RI序列的结构来研究Eco RI核酸内切酶中序列区分的结构基础。