ENGINEERING OF VARIANTS OF THE RESTRICTION-ENDONUCLEASE ECORV THAT DEPEND IN THEIR CLEAVAGE ACTIVITY ON THE FLEXIBILITY OF SEQUENCES FLANKING THE RECOGNITION SITE

The present work describes mutants of the restriction enzyme EcoRV tha t discriminate very efficiently between oligodeoxynucleotide substrate s with an EcoRV recognition sequence in different sequence context. Al l of these EcoRV variants harbor substitutions at position 226, where in the cocrystal structure of the specific EcoRV/DNA complex an argini ne contacts the backbone of the DNA substrate upstream of the recognit ion sequence, and cleave an oligodeoxynucleotide with an EcoRV site in a nonflexible sequence context (the recognition site being flanked by runs of A and T) with much higher catalytic efficiency (k(cat)/K-m) t han an oligodeoxynucleotide with an EcoRV site In a flexible sequence context (the recognition site being flanked by runs of AT), in contras t to the wild-type enzyme, that cleaves both substrates with the same catalytic efficiency. Steady-state and single-turnover kinetics indica te that the enhanced selectivity of the mutants is due to the catalyti c step of the reaction. It is possible to enhance the discriminatory p ower of these EcoRV variants through the choice of appropriate reactio n conditions, in particular low salt concentration and low reaction te mperatures. It must be emphasized that the enhanced selectivity of the se EcoRV variants toward EcoRV sites in a flexible and nonflexible seq uence context, respectively, is not only seen with oligodeoxynucleotid es, but also with plasmid substrates.