Random mutagenesis targeted to the active site of the EcoRV restriction endonuclease

Two segments of the gene for the EcoRV restriction endonuclease, each encoding 10 amino acids at the active site, were subjected to random mutagenesis with degenerate oligonucleotides. Mutations that abolished the activity of the EcoRV endonuclease were selected by viability in a strain of Escherichia coli that lacks the EcoRV methyltransferase, under conditions where the gene for the wild-type endonuclease is lethal to the cell. Sixty-five mutants were isolated and analyzed by DNA sequencing to identify the mutations. The collection of null mutants contained 49 with single amino acid substitutions, 15 with double substitutions, and one with a triple substitution. The single substitutions were located at many different positions within the two 10-amino acid segments, though several hot-spots gave rise to null mutants at high frequencies. Some hot-spots were readily explained by reference to the crystal structure of EcoRV since they were at the amino acids immediately adjacent to the scissile phosphodiester bond: for example, Asp90 and Lys92. These residues may be directly involved in the catalytic mechanism. Other hot-spots, such as Gln69, Tyr72, and Ala88, were at unexpected positions that appear to have no direct role in DNA binding or catalysis. At some of the unexpected hot-spots, the side chain of the amino acid lies distant from the DNA, yet the enzyme was still inactivated by conservative substitutions at these positions. The sensitivity of the EcoRV endonuclease to conservative substitutions may be due to its requirement to take up one particular conformation at the DNA-protein interface out of a large number of alternative conformations.