Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI.
BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward 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 downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), 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 R(1)R(2) 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 R(1)(-)R(2)(+) and R(1)(+)R(2)(-) 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(1) 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 R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.
Bellamy
Stuart R W
SR
Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol, BS8 1TD, UK. stuart.bellamy@bristol.ac.uk
Milsom
Susan E
SE
Scott
David J
DJ
Daniels
Lucy E
LE
Wilson
Geoffrey G
GG
Halford
Stephen E
SE
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
D021122:Protein Subunits; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C025629:GCAGC-specific type II deoxyribonucleases