PMC

Single-molecule relaxation by Topo I and Topo III. The plots show typical relaxation runs for different substrates. Each run is defined as a series of relaxation events where pausing cannot be observed. (A) Relaxation of negatively supercoiled DNA by Topo I. (B) Relaxation of positively supercoiled DNA with a 27-bp bulge by Topo I. (C) Relaxation of positively supercoiled DNA with 27-bp mismatch by Topo I. (D) Relaxation of negatively supercoiled DNA by Topo III. (E) Relaxation of positively supercoiled DNA with a 27-bp bulge by Topo III. (F) Relaxation of positively supercoiled DNA with 27-bp mismatch by Topo III. In all cases the plots show the length of the DNA plotted against time. Introduction of supercoils results in shortening of the molecule whereas relaxation results in elongation of the molecule. Negatively supercoiled and mismatched DNA experiments used a 0.7 pN stretching force; other experiments used a 2.0 pN force. Panel (D) illustrates the parameters used to analyze the relaxation events (see text for definitions). The gray trace corresponds to the measured events whereas the red trace corresponds to an unweighted running average over 10 events.

Relaxation rate per run and total relaxation rate for Topo I and Topo III. (A) Histogram showing the distribution of the relaxation rate per run. In all cases, Topo III has a faster relaxation rate per run than Topo I. In the case of negatively supercoiled DNA, Topo III is faster by a factor of ∼40. The inset shows the distribution of relaxation rate per run for negatively supercoiled DNA by Topo III. (B) Histogram showing the distribution of the total relaxation rate. Topo I has a faster total relaxation rate than Topo III, in agreement with bulk experiments. The inset shows the distribution of the total relaxation rate for negatively supercoiled DNA by Topo III. In all cases, shaded bars correspond to Topo I and white ones to Topo III. For mismatched substrates (marked by filled circles) Topo III relaxation was only observed in 20% of the experiments. The different substrates used are shown at the bottom, where -SC corresponds to negatively supercoiled DNA, and 12B, 12 M, 27B and 27 M correspond to the 12-bp and 27-bp bulged and mismatched substrates. In all cases, the differences observed between the two enzymes are significant as assessed by the P-value (P-value of 0.0001 for all compared pairs, except for the 12-bp mismatch in panel B, with a P-value of 0.02). The error bars shown correspond to the standard errors. Details on the number of events used for each histogram are found in Supplementary Table 1.

(A) Escherichia coli topoisomerase I relaxes negatively supercoiled DNA more efficiently than E. coli topoisomerase III in bulk experiments. The gel shows relaxation of negatively supercoiled DNA by either Topo I or Topo III. Identical mass amounts of protein and DNA were incubated for the same period of time and the resulting relaxed DNA products were analyzed in an ethidium bromide-stained agarose gel. The experiment shows that Topo I relaxes DNA more fully than comparable amounts of Topo III. The amount of protein used is shown at the top of the gel as well as the equivalent molarity for each lane. An amount of 200 ng (0.72 fmol) of DNA was used in each lane. R marks the position of relaxed DNA and SC the position of supercoiled DNA. (B) Substrates used for single-molecule experiments. Three different types of DNA molecules were employed for the single-molecule experiments: intact dsDNA (top), DNA with a single-stranded bubble or bulge (middle), and DNA with a mismatched region (bottom). The bulge and mismatch regions were used in experiments with positively supercoiled DNA as type IA topoisomerase require single-stranded regions for activity. The intact DNA molecules were used for experiments with negatively supercoiled DNA.

Characterization of DNA relaxation of different substrates by Topo I and Topo III. (A) Histogram showing the distribution of time lag before initiation of relaxation. In all cases, Topo III shows a much longer time lag before starting a relaxation run. For mismatched substrates (marked by filled circle) Topo III relaxation was only observed in 20% of the experiments. (B) Histogram of the secondary time lag. The P-values for the differences between the initial and secondary time lags are shown in Supplementary Table S2. In all cases, the differences between the two time lags are significant. (C) Histogram of the mean number of turns relaxed in a run (ΔLk). Topo III consistently removed more turns per run for all substrates. The inset shows the distribution for the 12-bp bulged substrate using Topo III. The solid curve corresponds to a fit of an exponential decay to the distribution. In all cases, shaded bars correspond to Topo I and white ones to Topo III. The different substrates used are shown at the bottom, where -SC corresponds to negatively supercoiled DNA, and 12B, 12 M, 27B and 27 M correspond to the 12-bp and 27-bp bulged and mismatched substrates. In all cases, the differences observed between the two enzymes are significant as assessed by the P-value (P-value of 0.0001 for all compared pairs, except for the 12-bp mismatch in panel A, with a P-value of 0.001). The error bars shown correspond to the standard errors. Details on the number of events used for each histogram are found in Supplementary Table S1.