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. 2017 Dec 14;46(2):861–872. doi: 10.1093/nar/gkx1247

Figure 3.

Figure 3.

Analysis of FI* and FI** DNA. (A) Generation of FI* and FI** over time during incubation with SsoTopA at 95°C. After incubation at 95°C for the indicated times, samples were cooled at 4°C for 5 min, centrifuged before adding 0.1% SDS, 25 mg/ml bromophenol blue and 15% sucrose (final concentrations). The samples were further incubated for an additional 5 min at 4°C. The samples were loaded onto an agarose gel at 4°C and run at 3 V/cm for 6 h at 4°C. (B) Spontaneous re-annealing of single-stranded forms FI** at 25°C. The samples were treated as in (A) except that they were further incubated for an additional 5 min at 25°C instead of 4°C prior to loading on the gel. (C) Sensitivity of the different DNA forms to S1 nuclease. Purified pTZ18R DNA obtained after incubation with TopA at 95°C for 4 min was further incubated at 4°C overnight without (0) or with 1 × 10−3 unit or 10 × 10−3 unit of S1 nuclease. (D) Sensitivity of the different DNA forms to T5 exonuclease. pTZ18R was linearized by BamHI (lanes 1–4) and then heated at 95°C for 5 min (lanes 2–4). Unlinked DNA forms (FI* and FI**) were obtained after incubation of pTZ18R with SsoTopA at 95°C for 5 min (lanes 6–8). After incubation at 95°C the DNA was further incubated at 4°C without (lanes 1, 2 and 6) or with 10 units of T5 exonuclease for 4 h (lanes 3 and 7) or 16 h (lanes 4 and 8). The SsoTopA treated samples (lanes 6–8) were additionally heated at 95°C for 2 min just before loading. Lane 5 is the control DNA substrate (pTZ18R). In the different experiments (A–D), the reaction products were analyzed by one dimensional gel electrophoresis: OC indicates open circular DNA, Lin the linear double stranded DNA, SSLin the linear single-stranded DNA, -SC the negatively supercoiled DNA form and the particular FI* and FI** forms.