title: DNA-protein interaction at the replication origins of plasmid chromosomes.
abstract: Novel techniques have been developed to purify replication initiator proteins of the plasmids R6K and pSC101. The techniques consist of tagging the initiator cistrons at the C-terminus with beta-galactosidase-encoding DNA of Escherichia coli in the correct translational phase. The hybrid proteins are then rapidly purified by adsorption to and elution from a beta-galactosidase- specific affinity column. Two procedures have been devised to isolate the nonfused initiator proteins using the fused protein as a handle. The first procedure, called subunit association chromatography, exploits the association of a monomer of nontagged protein with that of beta-galactosidase-tagged protein in isolating both types of proteins by beta-galactosidase specific affinity column chromatography. The second procedure involves the fusion of the initiator protein to beta-galactosidase via a specific linker DNA. The linker DNA encodes a protein which is readily and specifically hydrolyzed by a sequence specific protease, thus releasing the initiator protein from beta-galactosidase. Using purified or partially purified initiator protein, we have demonstrated that the R6K encoded initiator protein (Pi protein) binds to a consensus 22 bp sequence at 2 regions of the plasmid chromosome. The pSC101-encoded initiator protein binds to sequences at or near the plasmid replication origin. At low concentrations the protein binds to a nucleation site and upon raising the concentrations of the protein binding is promoted at 4 adjacent sequences that have partial homologies with the nucleation sequence. Deletion of the binding site leads to a nonfunctional replication origin.
authors.forename: D
authors.initials: D
authors.lastname: Bastia
authors.forename: C
authors.initials: C
authors.lastname: Vocke
authors.forename: J
authors.initials: J
authors.lastname: Germino
authors.forename: J
authors.initials: J
authors.lastname: Gray
mesh_terms: D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D010957:Plasmids; D011485:Protein Binding; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase