Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein.
Initiation of DNA replication from ori beta of plasmid R6K requires the presence of the ori gamma sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in gamma increases binding of the protein at the very weak binding site present in ori beta by cooperativity at a distance. The gamma-beta interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated gamma-beta interaction in vitro suggests that the cooperative interaction of gamma-bound protein with the beta sequence by DNA looping is an early step in the initiation of DNA replication at the beta origin of R6K.
Mukherjee
S
S
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Erickson
H
H
Bastia
D
D
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases; D011088:DNA Ligases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism* / Q000648:ultrastructure; D004270:DNA, Circular / Q000096:biosynthesis* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D008854:Microscopy, Electron; D008957:Models, Genetic; D011485:Protein Binding; D011815:R Factors; D013463:Sulfuric Acid Esters; D015534:Trans-Activators
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D013463:Sulfuric Acid Esters; D015534:Trans-Activators; C045264:replication initiator protein; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004265:DNA Helicases; D011088:DNA Ligases; C007482:dimethyl sulfate