Requirements for and regulation of origin opening of plasmid P1.
Origin opening is essential for the initiation of DNA replication in the theta mode and requires binding of initiator proteins. Using reactivity to KMnO4 in vivo as an assay, we find that, like initiation, origin opening of the Escherichia coli plasmid P1 requires the host initiators DnaA and HU and the plasmid-encoded initiator RepA. The ability to detect opening at the P1ori in vivo allowed us to study this activity at various copy numbers in chimeric replicons. The opening was prevented when the P1ori was cloned in high copy vectors or when excess RepA binding sites (iterons) were provided in trans. However, when RepA supply was also increased, the opening was efficient. A further increase in RepA prevented opening. Replication of an incoming P1 under these conditions correlated with opening. These results demonstrate that initiation is possible even at abnormally high origin concentrations and that oversupply of RepA, relative to iterons, can prevent replication by blocking origin opening. It appears that plasmid overreplication can be prevented either by limiting RepA or by accumulating RepA at a rate higher than that of the origin.
Park
K
K
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Mukhopadhyay
S
S
Chattoraj
D K
DK
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002874:Chromosome Mapping; D003090:Coliphages / Q000235:genetics; D018983:DNA Footprinting; D004265:DNA Helicases; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D008242:Lysogeny; D010957:Plasmids / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011196:Potassium Permanganate / Q000494:pharmacology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D012093:Replicon; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011506:Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C040330:histone-like protein HU, bacteria; C045264:replication initiator protein; D011196:Potassium Permanganate; D004265:DNA Helicases