Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.
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