Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system.
The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.
D002499:Centrifugation, Density Gradient / Q000379:methods; D002876:Chromosomes, Bacterial / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009587:Nitrogen Isotopes / Q000378:metabolism; D009693:Nucleic Acid Hybridization / Q000379:methods; D011815:R Factors / Q000378:metabolism*; D012093:Replicon / Q000502:physiology*; D013696:Temperature
D004269:DNA, Bacterial; D009587:Nitrogen Isotopes