The major long term objective of this research is to understand the detailed molecular mechanisms by which proteins discriminate and interact with specific DNA sequences. This problem will be approached through a detailed structural and functional analysis of the restriction endonuclease, EcoRI. John Rosenberg, U. of Pittsburgh, who has determined the structure of the EcoRI.DNA complex to 3A resolution, will collaborate in this effort. The primary aim of this proposal is a mutational analysis of EcoRI. The contribution of individual amino acids in sequence discrimination and catalysis will be investigated. Hydrogen bonds between the endonuclease and the major groove of the DNA ionic contacts with the phosphodiester backbone are critical components of these functions. The structural model will be used to target for mutagenesis amino acids which appear to be involved in these interactions. Altered proteins will be generated by cassette mutagenesis and mismatch oligonucleotide mutagenesis. Also selected region will be saturated with mutations utilizing degenerate oligonucleotides. Dr. Greene and co.workers developed a vector system so that mutants of this potentially lethal enzyme will be purified and characterized in order to determine the effects of individual amino acid substitutions on structure and on various functional parameters (e.g. binding energy, site discrimination, phosphodiester bond cleavage and editing). Crystallographic analysis of mutants will be carried out by Dr. Rosenberg.
