THE PRODUCTION AND CHARACTERIZATION OF ARTIFICIAL HETERODIMERS OF THERESTRICTION-ENDONUCLEASE ECORV

A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It wa s developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding differ ent EcoRV mutants are coexpressed in the same Escherichia coli cell le ading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transf erase or a His6-tag. The EcoRV heterodimer produced in vivo is separat ed from the two EcoRV homodimers and purified to homogeneity by affini ty chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdepen dence of the two catalytic centers, EcoRV heterodimers consisting of o ne subunit with wild type sequence and one subunit with amino acid sub stitutions in the PD...(D/E)XK motif, characteristic for the active si tes of many restriction endonucleases, were produced. While the homodi meric EcoRV active site mutants are catalytically inactive, the hetero dimeric EcoRV variants with one active and one inactive catalytic cent er display a twofold reduced activity toward oligodeoxynucleotide subs trates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzy me both catalytic centers function independently of each other.