Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease.
Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu----Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.
King
K
K
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Benkovic
S J
SJ
Modrich
P
P
D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping
D004269:DNA, Bacterial; D015246:Deoxyribonuclease EcoRI