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. 2018 Jun 9;46(13):6773–6784. doi: 10.1093/nar/gky470

Figure 1.

Figure 1.

DNA supercoiling by gyrase containing two or one CTD(s). (A) Time trace of ATP-dependent DNA supercoiling by gyrase with two CTDs and gyrase with one CTD (see cartoons of structures). 20 nM relaxed pUC18 was supercoiled by 40 nM GyrA and 160 nM GyrB in presence of 1.5 mM ATP at 37°C, and the reaction was stopped at indicated time points. Error bars depict the standard deviation from three independent experiments. Quantification of the fraction of supercoiled DNA as a function of time gives rate constants of supercoiling of ksc = 0.035 ± 0.011 s−1 for gyrase with both CTDs, and ksc = 0.020 ± 0.009 s−1 for gyrase with a single CTD. Errors are standard deviation from three independent experiments. Gyrase with one CTD thus catalyzes negative supercoiling of DNA only slightly more slowly than gyrase with two CTDs. (B) Concentration dependence of DNA supercoiling for gyrase with two, one, and no CTD(s). 20 nM relaxed pUC18 were incubated with 10, 20, 40, 80, or 160 nM GyrA and 20, 40, 80, 160, or 320 nM GyrB in the presence of 1.5 mM ATP, and reactions were stopped after 5 min. Compared to gyrase with two CTDs, about twice the concentration of gyrase with one CTD is needed to achieve complete supercoiling (red boxes). Gyrase lacking the CTDs (160 nM GyrAΔCTD, 320 nM GyrB) does not supercoil DNA. sc-: negatively supercoiled DNA, rel: relaxed DNA.