The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV.
A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.
Wende
W
W
Institut für Biochemie, Justus-Liebig-Universität Giessen, Germany.
Stahl
F
F
Pingoud
A
A
D001704:Biopolymers; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids
D001704:Biopolymers; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases