DNA-protein interaction at the replication origins of plasmid chromosomes.
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.
Bastia
D
D
Vocke
C
C
Germino
J
J
Gray
J
J
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*
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase