TOWARDS THE DESIGN OF RARE CUTTING RESTRICTION ENDONUCLEASES - USING DIRECTED EVOLUTION TO GENERATE VARIANTS OF ECORV DIFFERING IN THEIR SUBSTRATE-SPECIFICITY BY 2 ORDERS OF MAGNITUDE

The restriction endonuclease EcoRV cleaves DNA highly specifically wit hin GATATC sequences. In order to create EcoRV variants that have an e xtended recognition site we have employed a semi-rational random mutag enesis/selection procedure. Twenty-two amino acid residues were subjec ted to random mutagenesis and about 500 EcoRV variants representing th ree generations of mutants were screened. Among these some highly acti ve variants that strongly prefer AT-flanked cleavage sites (e.g. S183A /Q224R, T93S/I103F/S183A/T222S or N97T/S183A/T222S) and others that pr efer GC flanks (e.g. K104N/A181T) were identified. As wild-type EcoRV does not discriminate between those cleavage sites, the generation of these variants represents a significant first step towards redesigning EcoRV to become an 8 or 10 bp cutter. Such enzymes, only very rarely found in nature, could be extremely helpful for the manipulation of la rge DNA fragments. (C) 1998 Academic Press.