Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII.
EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.
Takahashi
Shuntaro
S
Frontier Research Center, Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama, Japan.
Matsuno
Hisao
H
Furusawa
Hiroyuki
H
Okahata
Yoshio
Y
D000494:Allosteric Regulation / Q000502:physiology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology
D029968:Escherichia coli Proteins; D004247:DNA; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific