Figure 3. Real-time Observation of Topoisomerase II-mediated DNA Cleavage/Religation Reaction.

(A,C) Representative time traces showing association/dissociation (black arrowheads) and bending/straightening events (red arrowheads) in the presence of 5 nM yTopoII and 10 mM Ca²⁺ (A) or Mg²⁺ (C) ions for Yclv1 DNA construct. In these measurements, a time resolution of 50 ms was used for observing transient interactions. Black arrowheads indicate transient binding of the enzyme to a DNA. Red arrowheads indicate the short-lasting bending events. Cy3 fluorescence, Cy5 fluorescence, and corresponding FRET efficiency are shown in green, red, and blue, respectively. Here, the total intensity is the sum of intensities of Cy3 and Cy5 and FRET efficiency (hereafter FRET) is the ratio of Cy5 intensity to the total intensity. To clearly visualize the enzyme binding events, red and yellow boxes are added as a visual guide. The same color convention is used throughout the paper.

(B,D) Representative time traces showing DNA cleavage/religation (red arrows) events in the presence of 5 nM yTopoII and 10 mM Ca²⁺ (B) or Mg²⁺ (D) ions. In (B), experiment was performed under the same reaction condition as in (A) except for the time resolution. In (D), the experimental condition was same as in (C) except for the time resolution. For long-time observation, a time resolution of 500 ms was used to reduce the photobleaching of the fluorophores. The long-lived cleavage events are indicated by red arrows.

(E,F) FRET histograms of Yclv1 DNA duplexes with bound enzyme in the presence of 5 nM yTopoII and 10 mM Ca²⁺ (E) or Mg²⁺ (F) ions. The cleavage population of Yclv1 (blue line, 83%) in the presence of Ca²⁺ ions was obtained by fitting the FRET histogram to the sum of three Gaussian functions.

(G,H) Relative distribution of total intensity and FRET efficiency of Yclv1 DNA duplexes in the presence of 5 nM yTopoII and 10 mM Ca²⁺ (G) or Mg²⁺ (H) ions. The colors represent the relative frequency of the events. White dashed lines were added for a clear visualization of the three species, including unbound DNA only, enzyme binding, and DNA bending/cleavage.