ECORI FUNCTION ASSESSED BY SITE-DIRECTED MUTAGENESIS

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We propose to coordinate structural, biochemical and genetic studies of the EcoRI endonuclease in order to assess the relationship of the structure of this protein to its functional mechanism. The specific aims of this research proposal are the introduction of defined mutations into the EcoRI endonuclease gene, analysis of the mutant endonucleases, and incorporation of the results of these analyses into a working structural model of the EcoRI endonuclease-substrate interaction. The mutational studies initially will focus on a 40 amino acid segment of the endonuclease which, at least in part, has been determined to be in direct contact with the DNA substrate. This region also includes an amino acid sequence which has been incorporated into a computergraphic developed working model for the recognition of the unique six-base pair DNA substrate of the EcoRI endonuclease. G greater than A and C greater than T transitional mutatations will be limited to the indicated region of the genome by selected bisulfite mutagenesis of the appropriate "deletion loop" of heteroduplex plasmid molecules. Other mutations will be introduced in this region by primer mismatched repair synthesis, and eventually by the development of "coding cassettes" for completely flexible designed mutational alterations. The endonuclease variants, define by nucleotide sequence determinations, will be analyzed for numerous functional parameters, e.g., stability of dimer structure, thermostability, kinetics of DNA hydrolysis, EcoRI* activity, recognition properties, binding equilibrium studies, points of contacts with DNA substrate, and assessment of electrostatic and non-electrostatic interactions. The results of these analyses will be referenced to the wild type endonuclease and the anticipated structural resolution of the EcoRI endonuclease-substrate complex to refine working models of the various steps of the EcoRI endonuclease mechanism. The latter effort will utilize the computergraphic facility at UCSF and the computergraphic modeling approach will be incorporated into the designing of specific mutational alterations for continued refinement of the molecular mechanism of the EcoRi endonuclease reaction with its substrate.