ACCURACY OF THE ECORI RESTRICTION ENDONUCLEASE - BINDING AND CLEAVAGE STUDIES WITH OLIGODEOXYNUCLEOTIDE SUBSTRATES CONTAINING DEGENERATE RECOGNITION SEQUENCES

We have synthesized a series of 18 nonpalindromic oligodeoxynucleotides that carry all possible base changes within the recognition sequence of EcoRl. These single strands can be combined with their complementary single strands to obtain all possible EcoRl* sequences (left), or they can be combined with a single strand containing the canonical sequence to obtain double strands with all possible mismatches within the recognition sequence (right): The rate of phosphodiester bond cleavage of these oligodeoxynucleotides by EcoRl was determined in single-turnover experiments under normal buffer conditions in order to find out to what extent the canonical recognition site can be distorted and yet serve as a substrate for EcoRl. Our results show that oligodeoxynucleotides containing mismatch base pairs are in general more readily attacked by EcoRl than oligodeoxynucleotides containing EcoRl* sites and that the rates of cleavage of the two complementary strands of degenerate oligodeoxynucleotides are quite different. We have also determined the affinities of these oligodeoxynucleotides to EcoRl. They are higher for oligodeoxynucleotides carrying a mismatch within the EcoRl recognition site than for oligodeoxynucleotides containing an EcoRl* site but otherwise do not correlate with the rate with which these oligodeoxynucleotides are cleaved by EcoRl. Our results allow details to be given for the probability of EcoRl making mistakes in cleaving DNA not only in its recognition sequence but also in sequences closely related to it. Due to the fact that the rates of cleavage in the two strands of a degenerate sequence generally are widely different, these mistakes are most likely not occuring in vivo, since nicked intermediates can be repaired by DNA ligase. © 1990, American Chemical Society. All rights reserved.