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: 6520360
• 负责人: JOHN M ROSENBERG
• 金额: $ 23.95万
• 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/6520360
• 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等广泛问题。该项目建议继续对Eco RI内切酶的结构和功能进行分析，Eco RI内切酶是一种识别六核苷酸GAATTC的限制酶。这一建议将X射线结晶学和分子动力学计算结合在一起，以协调的、基于结构的努力来理解Eco RI内切酶结构和功能之间的关系。主要目标是：1.研究Eco RI内切酶-DNA复合体，其中DNA包含碱基类似取代，这些取代扰乱蛋白质和DNA之间的特定接触，例如通过删除或修改DNA上的个别部分。X射线结晶学和分子动力学方法都将被使用。2.类似地研究Eco RI内切酶-DNA复合体，其中蛋白质包含改变特定蛋白质-DNA接触的定点突变。X射线结晶学和分子动力学方法都将被使用。3.研究了Eco RI位点两侧碱基对取代引起的已知结合能差异的结构基础。X射线结晶学和分子动力学方法都将被使用。4.通过确定含有错配或Eco RI序列的结构，探讨Eco RI内切酶序列识别的结构基础。