The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences

Abstract

It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5-bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase.