Relaxed plasmid DNA

Fig. 3. Unlinking and melting of covalently closed DNA by topoisomerase V. (A) Electrophoresis of control relaxed plasmid DNA (1), negatively supercoiled (2), and the same DNA as in (1) but after incubation at 95°C with topo V (3). —SC and U indicate negatively supercoiled and unlinked DNA, respectively. (B) Unlinked DNA strands of pBR322 DNA by electron microscopy. The molecule shown here is magnified approximately 120,000. ssDNA circles are linked onee. (C) An illustration of incomplete melting of circular DNA at high temperature and its complete melting and unlinking by topoisomerase V. Duplex regions and melted strands are shown by thick and thin lines.

Figure 12. Analytical anion-exchange HPLC of supercoiled and linearized plasmid DNA on a DEAE column. Sample, 0.19 supercoiled pUC8 DNA and 0.33 /tg pUC8 DNA linearized with icoRI column, Nucleogen DEAE-4000 (6 x 125 mm) apparatus, Merck-Hitachi 655 A-12 with a Meick-Hitachi 6S5A-22 spectrophotometer set at 260 nm with 0.08 AUFS elution, linear gradient from 0.66 to 1.2 M NaCI in 0.03 M sodium phosphate. pH 6.0, 5 M urea in 60 min flow-rate, 2 ml/min. Supercoiled plasmid DNA is eluted before (at 11.5 min) linear plasmid DNA (at 13.5 min). The minute peak following peak for supercoiled plasmid DNA represents relaxed plasmid DNA.

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