The EcoRI restriction endonuclease with bacteriophage λ DNA. Kinetic studies

Abstract

The kinetics of the reactions of the EcoRI restriction endonuclease at individual recognition sites on the DNA from bacteriophage lambda were found to differ markedly from site to site. Under certain conditions of pH and ionic strength, the rates for the cleavage of the DNA were the same at each recognition site. But under altered experimental conditions, different reaction rates were observed at each recognition site. These results are consistent with a mechanism in which the kinetic stability of the complex between the enzyme and the recognition site on the DNA differs among the sites, due to the effect of interactions between the enzyme and DNA sequences surrounding each recognition site upon the transition state of the reaction. Reactions at individual sites on a DNA molecule containing more than one recognition site were found to be independent of each other, thus excluding the possibility of a processive mechanism for the EcoRI enzyme. The consequences of these observations are discussed with regard to both DNA-protein interactions and to the application of restriction enzymes in the study of the structure of DNA molecules.