Author/Authors :
The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence، نويسنده , , thus: CCTCAGC/GCTGAGG?CC^TCAGC/GC^TGAGG. We show that R.BbvCI comprises two different subunits، نويسنده , , R1 and R2; that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn، نويسنده , , each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence، نويسنده , , nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R1 subunit remained functional nicked the bottom strand of the sequence، نويسنده , , producing CCTCAGC/GC^TGAGG، نويسنده , , while those in which the catalytic site in the R2 subunit remained functional nicked the top strand، نويسنده , , producing CC^TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair، نويسنده , , recombination، نويسنده , , and replication، نويسنده , , and for laboratory procedures that initiate from nicks، نويسنده , , such as DNA degradation، نويسنده , , synthesis، نويسنده , , and amplification.، نويسنده ,
Keywords :
DNA-nickase , type II restriction enzyme , asymmetric recognition sequence , heterodimer , catalytic-site mutagenesis
