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 is being approached through a detailed structural and functional analysis of the restriction endonuclease, EcoRI. Collaboration is taking place with John Rosenberg, U. of Pittsburgh, who has determined the structure of the EcoRI-DNA complex to 3A resolution. The primary aim of this proposal is a mutational analysis of EcoRI. The investigation concerns the contribution of individual amino acids in sequence discrimination and catalysis. Hydrogen bonds between the endonuclease and the major groove of the DNA and ionic contacts with the phosphodiester backbone are critical components of these functions. Structural models will be used to target for mutagenesis those amino acids which appear to be involved in these interactions. Altered proteins will be generated by cassette mutagenesis and mismatch oligonucleotide mutagenesis. A vector system has been developed so that mutants of this potentially lethal enzyme can be recovered and screened for altered phenotype. The most interesting mutant enzymes will be purified and characterized in order to determine the effects of individual amino acid substitutions on structure and on various functional parameters. Selected mutants will be subjected to reversion analysis. Crystallographic analysis of mutants will be carried out by Rosenberg.
