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 m utagenesis 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, un der conditions where the gene for the wild-type endonuclease is lethal to the cell. Sixty-five mutants were isolated and analyzed by DNA seq uencing to identify the mutations. The collection of null mutants cont ained 49 with single amino acid substitutions, 15 with double substitu tions, and one with a triple substitution. The single substitutions we re located at many different positions within the two 10-amino acid se gments, though several hot-spots gave rise to null mutants at high fre quencies. Some hot-spots were readily explained by reference to the cr ystal structure of EcoRV since they were at the amino acids immediatel y adjacent to the scissile phosphodiester bond: for example, Asp90 and Lys92. These residues may be directly involved in the catalytic mecha nism. Other hot-spots, such as Gln69, Tyr72, and Ala88, were at unexpe cted positions that appear to have no direct role in DNA binding or ca talysis. At some of the unexpected hot-spots, the side chain of the am ino acid lies distant from the DNA, yet the enzyme was still inactivat ed by conservative substitutions at these positions. The sensitivity o f the EcoRV endonuclease to conservative substitutions may be due to i ts requirement to take up one particular conformation at the DNA-prote in interface out of a large number of alternative conformations.