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Direct observation of topoisomerase IA gate dynamics

Nature Structural & Molecular Biology volume 25, pages 1111–1118 (2018)Cite this article

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Abstract

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA.

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Fig. 1: Proposed mechanism of type IA topoisomerase activity.
Fig. 2: Magnetic tweezers measurements of topoisomerase binding and gate opening.
Fig. 3: Force dependence of gate-opening dynamics.
Fig. 4: Three-state kinetics of topoisomerase IA gate dynamics.
Fig. 5: Molecular dynamics simulations of topo III gate opening.
Fig. 6: Kinetic scheme of type IA topoisomerase gate dynamics.

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Data availability

Source data for Fig. 4 are available with the paper online. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health (HL001056–07 to K.C.N.) and a grant from the National Institutes of Health (R01GM054226 to Y.-C.T.-D.). This work used the computational resources of the NIH HPC Biowulf cluster. We thank Y. Seol (National Heart, Lung, and Blood Institute, National Institutes of Health) for insightful discussions, for experimental assistance, and for providing the DNA substrates. We thank L. Bradley (National Heart, Lung, and Blood Institute, National Institutes of Health) for assistance with purifying topoisomerase III and B. Cheng (New York Medical College) for purified topoisomerase I.

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Authors and Affiliations

  1. Laboratory of Single Molecule Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

    Maria Mills & Keir C. Neuman

  2. Biomolecular Sciences Institute, Florida International University, Miami, FL, USA

    Yuk-Ching Tse-Dinh

  3. Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA

    Yuk-Ching Tse-Dinh

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Contributions

M.M. and K.C.N. conceived the experiments. M.M. conducted the experiments and simulations and analyzed the data. Y.-C.T.-D. provided materials. M.M., K.C.N., and Y.-C.T.-D. wrote the manuscript.

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Correspondence to Keir C. Neuman.

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Supplementary Figure 1 Hairpin-gate-opening analysis.

(a) Comparison of DNA extension of an individual tether before (left) and after (right) the addition of 500 pM topo I. Black lines are DNA extension, blue lines are applied force (9–24 pN). Extension increases are above the baseline extension of the tether in the absence of protein. (b) Histograms of DNA extension of the same tether under force before and after addition of protein. (c-d) Examples of multi-Gaussian fits to a single unfolding cycle for topo III (c) and topo I (d). Histograms of smoothed data are in red, Residuals are shown in the top graph, individual Gaussians in the bottom graph. The overall fit (blue line) is shown overlaid on the histograms in the center graph. Inset in (c) shows an example of smoothed data for two events (black lines). Raw data is shown in gray. (e-f) Histograms of gate opening extension increases for topo III (ntethers = 4, ncycles = 58) (e) and topo I (ntethers = 5, ncycles = 25) (f). Error bars are s.e.m. Black lines are Gaussian fits. (g) Average survival times for tethers in the presence of 1 % SDS. Note that the time scale is logarithmic. Data was combined for topo I (ntethers = 3) and topo III (ntethers = 2). Average lifetime of tethers with evidence of cleavage was 27.7 ± 5.7 s. Average lifetime of the DNA tethers alone in 1 % SDS was 1486 ± 663 s. Error bars are s.d

Supplementary Figure 2 Extension-change distributions of the gapped DNA substrate.

Distance distributions (red) for the DNA extension change measured with the gapped DNA substrate for topo III at 8, 10, 12, 14 and 16 pN (ntethers = 8) and topo I at 12, 14, 16, 18 pN (ntethers = 7). Bin size is 0.5 nm. Blue lines are Gaussian fits obtained using the Igor multipeak fitting algorithm. Error bars are s.e.m. KD and extension change values are reported in Supplementary Table 1

Supplementary Figure 3 Closed-state lifetimes.

(a) Histograms of topo III short-lived closed state lifetimes (t ≤ 20 s), bin size 0.2–0.5 s, ntethers = 8. Data were fit to single exponentials (black) to determine kopen. Reduced χ2 values from exponential fits are 0.76 (8 pN), 1 (10 pN), 0.74 (12 pN), 1.2 (14 pN), and 1.1 (16 pN). (b) Histograms of topo III long-lived closed state lifetimes (t > 20 s), bin size 10–50 s, ntethers = 8. Data were fit to single exponentials (black) to determine kcleavage. Reduced χ2 values from exponential fits are 0.58 (8 pN), 0.5 (10 pN), 0.11 (12 pN), 0.36 (14 pN), and 0.76 (16 pN). (c) Histograms of topo I closed state lifetimes (bin size 0.1–0.2 s), ntethers = 7. Black lines are double-exponential fits. Reduced χ2 values are 1.1 (12 pN), 1.1 (14 pN), 0.56 (16 pN), and 0.72 (18 pN). Gray lines are single exponential fits. Reduced χ2 values are 1.6 (12 pN), 2.4 (14 pN), 1.1 for (16 pN), and 1.5 (18 pN). Comparison of these fits using an F-test resulted in F values of 8.9 for (12 pN), p = 0.00093, 22.2 (14 pN), p < 0.00001, 13.6 (16 pN), p = 0.00011, and 30.0 (18 pN), p < 0.00001. The faster rate was assumed to be kopen and the slower rate to be 1/(tcleavage + topen). Rates obtained from the fits are shown in Supplementary Table 2. Error bars are s.e.m

Supplementary Figure 4 Open-state-lifetime distributions.

(a) Histograms of topo III open state lifetimes, bin size 2–5 s, ntethers = 8. Exponential fits in black. Reduced χ2 values are 0.22 (8 pN), 1.1 (10 pN), 1.5 (12 pN), 0.56 (14 pN), and 1.3 (16 pN). (b) Histograms of topo I open state, bin size 0.2–0.5 s, ntethers = 7. Exponential fits are shown in black. Reduced χ2 values are 2.7 (12 pN), 1.5 (14 pN), 2.7 (16 pN), and 1.9 (18 pN). Rates from the fits are shown in Supplementary Table 2. Error bars are s.e.m

Supplementary Figure 5 Effects of magnesium on topo-DNA binding.

(a) Average number of binding events (solid bars) and gate opening events (striped bars) from hairpin opening and refolding experiments in the presence (topo III ntethers = 4, ncycles = 58, topo I ntethers = 5, ncycles = 25) and absence of added magnesium. Error bars are s.d. In the absence of magnesium (topo III ntethers = 2, ncycles = 18, topo I ntethers = 4, ncycles = 8), less than half the number of bound proteins was observed for both topo III and topo I, whereas the ratio of bound proteins to opening events remained constant. For 0 Mg2+ results, only tethers for which at least one binding event was observed were counted, resulting in a possible over-estimation of the average number of binding events. (b) Example extension as a function of time at 14 pN for topo I at 3 mM Mg2+ and 0 mM Mg2+ with EDTA. Religation is reduced but not eliminated even under chelating conditions

Supplementary Figure 6 Details of free-energy calculations from simulations.

(a-b) Force dependent free energies. The equilibrium free energy calculated from the simulations is shown in black, along with ΔGF curves for F = 8 pN (dark gray), F = 12 pN (medium gray), F = 16 pN (light gray). (a) Force-dependent free energy profile in which the entire free energy profile is force dependent. (b) Force-dependent free energy in which the initial opening depends on a force-independent conformational change. (c) Heat map of a two-dimensional free energy profile along the restrained umbrella sampling reaction coordinate, the distance between the centers of mass of domains III and IV, and x, the distance between the catalytic tyrosine and the DNA cleavage site. Two parallel pathways can be observed, one in which the decatenation loop forms contacts with an acidic loop in domain II, and one in which the decatenation loop makes no contacts with the rest of the protein. (d) Structures of open state without decatenation latch. In the absence of latch formation, the hinge region of domain II adopts a more extended conformation. (e) Open structure of topo III from simulations (cyan) aligned with the structure of an isolated fragment of topo I consisting of domains II and III (purple, PDBID: 1CYY, chain A). The predicted structure of domain II in the open state based on the fragment conformation is similar to the simulation structure. (f) Structural alignment rotated to show differences in curvature of the β sheet

Supplementary Figure 7 Negative-supercoil relaxation by topo I and topo III at high force.

(a) Schematic of high force relaxation experiments (not to scale). DNA was negatively supercoiled at low force (F < 0.5 pN). The force was then raised to 12 pN for 5 to 15 min, after which is was lowered again. Relaxation was observed as a change in the linking number inferred from the change in extension after decreasing the force. The low force extension was compared with the original low force extension as a function of linking number curve measured in (a). (b) Examples of high force relaxation events for topo III and topo I. Blue lines represent applied force, black lines are DNA extension. Green arrows indicate rotation of magnets. Before and after hat curves are also shown. The fact that the molecules could be supercoiled after relaxation demonstrates that they were relaxed by the topoisomerase and not nicked, whereas the shift in the center of the extension versus magnet rotation curves verifies that the negative supercoiling was relaxed by the topo IA enzyme. Both proteins required more time to relax DNA at 12 pN than at forces below 0.5 pN

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Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Notes 1 and 2, and Supplementary Tables 1–4

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Supplementary Video 1

Gate dynamics from MD simulations. Changes in the topo III structure over the course of the umbrella sampling simulations. Domain I is shown in red and domain IV in blue. The black dashed line indicates the distance between the catalytic tyrosine (green) and the cleavage site on the DNA backbone

Supplementary Video 2

Interactions between decatenation loop and acidic loop from simulations. The decatenation loop is shown in blue and the acidic loop in red. Key residues are indicated

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Mills, M., Tse-Dinh, YC. & Neuman, K.C. Direct observation of topoisomerase IA gate dynamics. Nat Struct Mol Biol 25, 1111–1118 (2018). https://doi.org/10.1038/s41594-018-0158-x

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