Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.
We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.
Miron
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Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
Mukherjee
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Bastia
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D001345:Autoradiography; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D016172:DNA Fingerprinting; D004265:DNA Helicases; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity; D015534:Trans-Activators
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C499290:OppA protein, E coli; D015534:Trans-Activators; C046506:oligopeptide-binding protein, bacteria; C045264:replication initiator protein; D004265:DNA Helicases