Sequence context influencing cleavage activity of the K130E mutant of the restriction endonuclease EcoRI identified by a site selection assay

We have generated several EcoRI mutants which exhibit a decreased cleavage rate on one of the five specific cleavage sites in bacteriophage lambda-DNA. To study the influence of the sequence context on the cleavage rate in more derail, we developed a site selection assay. From a complete set of 4096 plasmid substrates, differing in three bases on both sides of a recognition sequence, optimal (best cut) and unfavorable (worst cut) sequences were selected by repeated limited digestion, separation, and in vivo amplification of cleaved and uncleaved plasmids. In order to compare the sequence preferences of the inner arm mutant K130E and the wild type enzyme, the cleavage rates and sequences of individual plasmids from the resulting pools were determined. The inner arm mutant K130E selected pools with clearly defined consensus sequences and a high amount of palindromic sequences. The cleavage rates of the selected sequences are specific for the K130E mutant as is shown by their cleavage with other mutants. In contrast, wild type EcoRI does not lead to a selection in this assay. Pre-steady state kinetics show that preferences for a certain sequence context are a result. of differences in the dissociation rates of the wild type enzyme. EcoRI is evolved to efficiently recognize and cleave each nonmethylated DNA invading the cell. Therefore, a fast dissociation after cleavage is not mandatory.