SEQUENCE CONTEXT INFLUENCING CLEAVAGE ACTIVITY OF THE K130E MUTANT OFTHE RESTRICTION-ENDONUCLEASE ECORI IDENTIFIED BY A SITE SELECTION ASSAY

We have generated several EcoRI mutants which exhibit a decreased clea vage rate on one of the five specific cleavage sites in bacteriophage lambda-DNA. To study the influence of the sequence context on the clea vage rate in more derail, we developed a site selection assay. From a complete set of 4096 plasmid substrates, differing in three bases on b oth sides of a recognition sequence, optimal (best cut) and unfavorabl e (worst cut) sequences were selected by repeated limited digestion, s eparation, 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 i ndividual plasmids from the resulting pools were determined. The inner arm mutant K130E selected pools with clearly defined consensus sequen ces and a high amount of palindromic sequences. The cleavage rates of the selected sequences are specific for the K130E mutant as is shown b y 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 diff erences in the dissociation rates of the wild type enzyme. EcoRI is ev olved to efficiently recognize and cleave each nonmethylated DNA invad ing the cell. Therefore, a fast dissociation after cleavage is not man datory.