Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI

The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCGAGG -> CC boolean AND TCAGC/GC boolean AND TGAGG. We show that R.BbvCI comprises two different subunits, R-1 and R-2; that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one bite remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R-1 subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC boolean AND TGAGG, while those in which the catalytic site in the R-2 subunit remained functional nicked the top strand, producing CC boolean AND TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification. (c) 2005 Elsevier Ltd. All rights reserved.