STRUCTURAL BASIS OF ECO RI ENDONUCLEASE SPECIFICITY

本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，以及解决酶-底物识别和特异性的分子基础的广泛问题。 本项目建议继续对识别六核苷酸GAATTC的限制性内切酶EcoRI进行结构-功能分析。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，基于结构的努力，以了解ECO RI内切核酸酶的结构和功能之间的关系。 主要目标是：1。 研究EcoRI核酸内切酶-DNA复合物，其中DNA含有干扰蛋白质和DNA之间特异性接触的碱基类似物取代，例如通过删除或修饰DNA上的单个部分。将采用X射线晶体学和分子动力学方法。2. 类似地研究EcoRI核酸内切酶-DNA复合物，其中蛋白质含有改变特定蛋白质-DNA接触的定点突变。 将采用X射线晶体学和分子动力学方法。3. 研究了由紧邻EcoRI位点侧翼的碱基对中的取代引起的结合能的已知差异的结构基础。 将采用X射线晶体学和分子动力学方法。4. 通过确定含有错配序列或EcoRI序列的结构，研究EcoRI核酸内切酶序列鉴别的结构基础。

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.