STRUCTURAL BASIS OF ECO RI ENDONUCLEASE SPECIFICITY

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

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.