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Comparative Study
. 2013 Apr;41(8):4640-9.
doi: 10.1093/nar/gkt136. Epub 2013 Mar 4.

Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification

Yeonee Seol  1 , Ashley H Hardin, Marie-Paule Strub, Gilles Charvin, Keir C Neuman
Affiliations
Comparative Study

Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification

Yeonee Seol et al. Nucleic Acids Res. 2013 Apr.

Abstract

Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed. The kinetic proofreading (KPR) model postulates that strand passage requires a DNA-bound topoisomerase to collide twice in rapid succession with a second DNA segment, implying a quadratic relationship between DNA collision frequency and relaxation rate. To test this model, we used a single-molecule assay to measure the unlinking rate as a function of DNA collision frequency for Escherichia coli topoisomerase IV (topo IV) that displays efficient non-equilibrium topology simplification activity, and for E. coli topoisomerase III (topo III), a type IA topoisomerase that unlinks and unknots DNA to equilibrium levels. Contrary to the predictions of the KPR model, topo IV and topo III unlinking rates were linearly related to the DNA collision frequency. Furthermore, topo III exhibited decatenation activity comparable with that of topo IV, supporting proposed roles for topo III in DNA segregation. This study enables us to rule out the KPR model for non-equilibrium topology simplification. More generally, we establish an experimental approach to systematically control DNA collision frequency.

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Figures

Figure 1.
Figure 1.
Topoisomerase unlinking assay and KPR model. (A) Cartoon of experiment (not to scale). A 1-µm magnetic bead (beige) is tethered to the surface of the flow chamber by two 5 kb torsionally unconstrained dsDNA molecules (blue and gold). A link between the DNA molecules is generated by rotating the bead (black arrow) using a magnet assembly (not shown), which decreases the DNA extension by ΔZ (red arrow). Strand passage mediated by a topoisomerase (shown bound to DNA), unlinks the DNA (blue arrow), leading to an increase in DNA extension. After a brief re-equilibration time, the process is automatically repeated. Extension of the double tether as a function of time shows the decrease in extension associated with introducing the link, and the subsequent increase in extension when the link is resolved by the strand-passage reaction of the topoisomerase. The time between these events is defined as the waiting time (Twait). DNA extension was measured by video tracking the bead in real time at 100 Hz with ∼2-nm spatial resolution. (B) DNA strand-passage mechanisms. DNA unlinking can occur via a single- or double-collision mechanism. Topoisomerase binds the G-segment (blue dot 1) and collides with a potential T-segment with rate constant kc (blue dot 2). In the single-collision mechanism (blue shaded arrow), strand passage occurs from this state with rate constant kuL. In the double-collision mechanism, i.e. the KPR model (pink shaded arrow), the initial T-segment collision activates the topoisomerase (yellow dot 1*). The activated topoisomerase will effectuate strand passage if it collides with a second potential T-segment (yellow dot 2*) before it decays back to the inactive state (blue dot 1) with rate constant kd. The single-collision strand-passage rate (RUL) is proportional to the collision frequency kc, whereas the double-collision RUL, posited by the KPR model, is proportional to the square of the collision frequency. Here, kon and koff are on and off rate constants of topoisomerase binding to G-segment DNA. kr and kr′ are release rate constants of a potential T-segment by the topoisomerase.
Figure 2.
Figure 2.
Characterization of DNA tether geometry and rotation-dependent collision frequency obtained from MC simulations. (A) In situ measurement of DNA tether geometry (not to scale). The double tether consists of two 5-kb DNA molecules attached between the surface and a streptavidin-coated magnetic bead (grey). To obtain the tether geometry, the DNA extension was measured as the bead was rotated from −0.5 to 0.5 rotations in increments of 0.1 turns (blue dots and dashed line). The DNA extension over the range −0.5–0.5 rotations was fit with Equation (1) (black solid line) to obtain the spacing between the DNA molecules, 2e, and the DNA extension at zero rotation, Z0. (B) Example collision frequency distributions for different imposed turns, n, from MC simulations of one double tether. Distributions of collision frequencies (number per 103 simulation steps) are fit with Gaussians (black solid lines) to obtain the mean collision frequency, <formula image>, for each turn. (C) Mean collision frequency, <formula image>, as a function of imposed turns, n. Values are colour-coded based on the corresponding number of imposed turns shown in B.
Figure 3.
Figure 3.
DNA extension traces of unlinking and mean rates of unlinking as a function of bead rotation. (A) DNA extension traces show unlinking activity of topo IV at three different imposed bead rotations (0.6, 0.8 and 1) for one double tether. The distributions of waiting times (Twait) (right panels) are well fit by single exponentials (black solid lines) to obtain the unlinking rates, k(n). (B) The unlinking rates are plotted as a function of bead rotation, n. Error bars correspond to the SD of the fit parameter.
Figure 4.
Figure 4.
Normalized unlinking rates of topo IV as a function of bead rotation and normalized collision frequency. (A) Normalized unlinking rates of topo IV measured with 12 different double tethers, formula image, are plotted as a function of bead rotation (n) for applied forces of 1 pN (triangles) and 2 pN (circles). Error bars are calculated through propagation of uncertainties (SD) in the initial fit parameters. (B) formula image as a function of the normalized simulated collision frequency, formula image, fit with linear (solid line), quadratic (dashed line) and power (short-long dashed line) functions. The power fit, formula image returned a power close to 1 (A = 0.90 ± 0.01 and P = 1.10 ± 0.03, formula image). The data are poorly fit by a quadratic relation (formula image) as evidenced by the 2-fold increase in reduced chi-square, formula image. Fitting to a line returned a slope of 0.87 ± 0.01 and a linear correlation coefficient of 0.75 with formula image.
Figure 5.
Figure 5.
Normalized unlinking rates of topo III versus normalized collision frequency and comparison with topo IV. (A) Normalized unlinking rates of topo III, formula image are plotted as a function of the normalized collision frequency from MC simulations, formula image, and fit to a line, with slope = 1.00 ± 0.01, formula image, and a linear correlation coefficient = 0.76. Error bars are calculated through propagation of uncertainties (SD) in the initial fit parameters. (B) Normalized rates of topo IV are directly compared with the normalized rates of topo III in a scatter plot, in which each point corresponds to the two normalized unlinking rates (topo III -x, and topo IV -y) measured with double tethers of comparable geometry at the same imposed rotation. Fitting the scatter plot with a linear function gave a slope S = 0.97 ± 0.02 with linear correlation coefficient 0.78 and formula image. Fitting to power function (y = AxP) returned A = 0.99 ± 0.04 and P = 1.02 ± 0.05 with formula image. Fitting to a quadratic function (y = Ax2) resulted in a significantly increased reduced chi-squared value, formula image.
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References

    1. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 2001;70:369–413. - PubMed
    1. Corbett KD, Berger JM. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 2004;33:95–118. - PubMed
    1. Forterre P, Gribaldo S, Gadelle D, Serre MC. Origin and evolution of DNA topoisomerases. Biochimie. 2007;89:427–446. - PubMed
    1. Schoeffler AJ, Berger JM. DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q. Rev. Biophys. 2008;41:41–101. - PubMed
    1. Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 2002;3:430–440. - PubMed

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