Figure 2. Force and concentration dependence of the rate at which ScTopA initiates DNA relaxation. (A) The initial time lag (ITL) for ScTopA activity displays.

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Figure 2. Force and concentration dependence of the rate at which ScTopA initiates DNA relaxation. (A) The initial time lag (ITL) for ScTopA activity displays a single-exponential distribution and decreases with increasing extending force. Histograms show the distribution of ITL values measured for 5-nM ScTopA and DNA extended by a force F = 0.5 pN (main panel) compared to ITL values measured at low extending force (inset, F = 0.3 pN). The lower extending force resulted in a near abolition of DNA relaxation events and a dramatic elevation of the number of unrelaxed molecules (grey bars in both panels). The number of molecules analysed (n) is shown at the top of each panel. The line corresponds to a single-exponential fit to the data obtained at 5 nM and for which unrelaxed molecules are proportionally very infrequent, giving a mean ITL = 29 s. Dashed vertical bars indicate the fraction of molecules relaxed within 100 s and 800 s, respectively. (B) ScTopA activity initiates more rapidly as concentration increases. The rate of initiation 1/ITL is obtained at each concentration by fitting a single-exponential decay to the cumulative probability distribution of individual ITLs. The cumulative probability distribution allows one to include in the analysis the unrelaxed DNA molecules (which become more frequent at lower concentrations), and by fitting at short timescales the characteristic decay time of the distribution is obtained. For 0.5 nM, n = 43 of which 15 are molecules unrelaxed after 800 s; for 1 nM, n = 39 of which 12 are unrelaxed molecules; for 1 nM, n = 59 of which four are unrelaxed molecules; statistics for 5 nM are given in (A); and for 10 nM, n = 42 of which one is an unrelaxed molecule. Error bars represent SEM obtained from the single-exponential fit. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. ScTopA concentrations do not influence enzyme processivity Figure 3. ScTopA concentrations do not influence enzyme processivity. The enzyme processivity as expressed by the ratio between initial supercoiling (IS) and initial relaxation (IR). Each circle represents a single DNA relaxation event; circles accumulated near the dotted line correspond to nearly full relaxation of supercoiled DNA in single relaxation event (IS = IR), circles marked with a ellipse represent the fraction of DNA molecules remaining supercoiled (IR = 0 within the 800-s recording time); circles located in between correspond to molecules relaxed in more than one burst (IS > IR). Percentage of molecules belonging to each population are shown on each diagram in brackets. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 1. Single-molecule assay for analysis of ScTopA relaxation activity. (A) The scheme of the experiment. (B) Extension versus supercoiling curve for 2-kb dsDNA extended by a force F = 0.3 pN. (C) Time-trace of ScTopA activity acting on 2-kb dsDNA for positive (+SC) and negative (−SC) supercoiling. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. Relaxation bursts and pausing by ScTopA Figure 4. Relaxation bursts and pausing by ScTopA. (A) The number of relaxation bursts required to remove negative supercoils from DNA is not dependent on the length of DNA substrates. Histograms show the fractions of 2–51-kb DNA molecules relaxed in the specified number of bursts (1–5). (B) Example of a pause between two successive relaxation bursts and distribution of pause lifetimes. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. ScTopA relaxation exhibits high velocity and processivity of supercoils removal (L<sub>k</sub>/s) in a single burst. (A) Typical time-trace for supercoils relaxation as observed on 51-kb DNA substrate supercoiled by −150 turns. (B) Burst velocity distribution and (inset) expanded view of a relaxation burst. (C) Burst processivity distribution. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. Residual supercoiling left unremoved by ScTopA relaxation bursts indicates ScTopA switches between high and low processivity as a function of torque acting on the DNA. Seventeen-kilobase DNA supercoiled by −50 turns and left to react with TopA shows (A) relaxation of ∼38 supercoils in the first 250 s of incubation but only (B) relaxation of ∼3 additional supercoils in the following 750 s of the reaction, for an extending force of about 0.3 pN. (Inset) When the extending force is increased to 1 pN, the average residual supercoiling is zero after a 250-s reaction. (C) Fifty-one-kilobase DNA supercoiled by −150 turns and left to react with TopA for 250 s shows an increase in residual supercoils roughly three times greater than what is observed on 17-kb DNA. From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 7. The relaxation of negatively supercoiled pUC19 plasmid by ScTopA is dependent on its C-terminal domain. (A) Agarose gel-based assay demonstrating rapid appearance of low-density topoisomers produced by ScTopA even at low protein concentration (25–50 nM; lanes 1 and 2). (B) Comparison of processive and distributive removal of supercoils during the reaction sustained by wild-type ScTopA (100 nM) and mutant ScTopA881 truncated in C-terminus by 71 amino acids (100 nM), respectively. (C) Estimation of kinetic parameters V<sub>max</sub> and K<sub>m</sub> by fitting to the Michaelis–Menten equation for wild-type ScTopA and mutant ScTopA881. (D) Deletion of C-terminal domain (343 aa) completely abolishes the activity of the ScTopAΔC mutant (700 nM). From: A highly processive topoisomerase I: studies at the single-molecule level Nucleic Acids Res. 2014;42(12):7935-7946. doi:10.1093/nar/gku494 Nucleic Acids Res | © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com