Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI

BbvCI cleaves an asymmetric DNA sequence, 5'-CC down arrow TCAGC-3'/5'-GC down arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R-1, which acts at GC down arrow TGAGG; and R-2, which acts at CC down arrow TCAGC. Some mutants of BbvCI with defects in one subunit either R1-R2+ or R1+R2-, cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R1R2 heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R1-R2+ and R1+R2- mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R, subunit. The other strand is then cleaved slowly by R-2 before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R-1(+) and R-2(+) subunits in the wild-type enzyme can differ from their activities in the R1+R2- and R1-R2+ mutants. Each active site in BbvCI therefore influences the other. (c) 2005 Elsevier Ltd. All rights reserved.