Cleavage of Individual DNA Strands by the Different Subunits of the Heterodimeric Restriction Endonuclease BbvCI

BbvCI cleaves an asymmetric DNA sequence, 5′-CC↓TCAGC-3′/5′-GC↓TGAGG-3′, as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R1, which acts at GC↓TGAGG; and R2, which acts at CC↓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 R1 subunit. The other strand is then cleaved slowly by R2 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 R1+ and R2+ 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.