Conformation of the origin of P1 plasmid replication. Initiator protein induced wrapping and intrinsic unstacking.
The origin of plasmid DNA replication in bacteriophage P1 has five 19 base-pair sites that bind the plasmid-encoded initiator, RepA. Here we show, using a DNA band retardation assay, that RepA can bend DNA that carries one or more of the RepA binding sites. RepA binding to supercoiled DNA carrying the five sites, directly repeated and phased two turns of B-DNA apart, absorbs about one positive superhelical turn of DNA as determined by two-dimensional gel electrophoresis. This indicates that the DNA is wrapped around RepA as a consequence of in-phase bending at the individual binding sites. The RepA-DNA complexes did not show elevated sensitivity to KMnO4, a reagent specific for pyrimidine bases (T >> C) in unstacked DNA. The wrapping of the DNA around RepA, therefore, does not lead to significant unwinding of the double helix. Extensive unwinding, suitable for the initiation of DNA replication, most likely requires participation of factors other than RepA. We also noted that the thymine bases of the sequence 5'-ATC-3', of which there are 20 in the origin, all reacted to KMnO4 strongly whether or not RepA was present. The preferential reactivity of ATC sequences was specific to the origin region, as thymine residues including those in the ATC sequences did not display elevated sensitivity to KMnO4 in a DNA fragment from pBR322. On one of two strands of the origin the selective reactivity at the ATC sequences was supercoiling dependent. These results indicate that the origin includes unstacked DNA bases, the significance of which remains to be determined.
Laboratory of Biochemistry, NCI, NIH, Bethesda, MD 20892.
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Mukhopadhyay
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DK
Chattoraj
D001426:Bacterial Proteins / Q000378:metabolism; D017101:Bacteriophage P1 / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011506:Proteins; D015534:Trans-Activators
D001426:Bacterial Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D003850:Deoxyribonuclease I; D004265:DNA Helicases