Figure 1. Removal of the 2B subdomain activates Rep monomer unwinding

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Figure 1. Removal of the 2B subdomain activates Rep monomer unwinding Figure 1. Removal of the 2B subdomain activates Rep monomer unwinding. (A) wtRep structure in the ‘closed’ conformation ... Figure 1. Removal of the 2B subdomain activates Rep monomer unwinding. (A) wtRep structure in the ‘closed’ conformation (PDB 1uaa) showing its four subdomains (left), and a structural model of RepΔ2B with 2B replaced by three glycine residues (right). (B) Schematic of SF1 helicase (here, wtRep) monomer activity on DNA. 3′-to-5′ translocation on the 3′-strand of a DNA fork leads to unwinding of the duplex (left) with 2B in the closed state. 3′-to-5′ translocation on the 5′-strand leads to duplex rezipping behind the helicase (right) with 2B in the open state. (C) Schematic of dual-optical trap measurement of DNA hairpin unwinding. Two beads (grey spheres) held in traps (orange cones) stretch a DNA hairpin (blue) at constant tension via biotin-streptavidin (black square-yellow cross) and digoxigenin-anti-digoxigenin (yellow pentagon-black rectangle) linkages. Unwinding of a hairpin base pair releases two nucleotides, increasing DNA extension. (D) Representative data trace of DNA hairpin unwinding by wtRep dimer (schematic, top left corner). Close-up (inset) of one round of activity showing unwinding (U) followed by hairpin rezipping (Z). (E) Representative data trace of processive unwinding of DNA hairpin by RepΔ2B monomer (schematic, top left corner). The close-up (inset) of two rounds of activity shows hairpin unwinding (U) and rezipping (Z). The grey horizontal dashed line (D–E) indicates the limit expected for complete hairpin opening. (F) Schematic of dual-optical trap measurement of DNA fork (magenta) unwinding. Unwinding of a single base pair of the fork releases one nucleotide, changing DNA extension. (G) Representative data trace of processive DNA fork unwinding by RepΔ2B monomer (schematic, top left corner). The close-up (inset) shows two rounds of activity; protein unwinding (U) ends either by mid-fork dissociation (D) or dissociation at the base of the fork after three rounds of unwinding (U) and rezipping (Z). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Nucleic Acids Res, Volume 47, Issue 5, 23 January 2019, Pages 2523–2532, https://doi.org/10.1093/nar/gkz023 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Effect of duplex stability on unwinding processivity of RepΔ2B and wtRep. (A–C) Representative traces of ... Figure 2. Effect of duplex stability on unwinding processivity of RepΔ2B and wtRep. (A–C) Representative traces of hairpin DNA unwinding by wtRep dimer (A, shades of dark blue), hairpin DNA unwinding by RepΔ2B monomer (B, shades of blue), and fork DNA unwinding by RepΔ2B monomer (C, shades of magenta) showing unwinding processivity with increasing force. Values in units of k<sub>B</sub>T correspond to destabilization energies (see SI Text). (D) Median processivity as a function of destabilization energy for wtRep hairpin (open dark blue circles, N = 37–67), RepΔ2B hairpin (filled blue circles, N = 34–770), and RepΔ2B fork (filled magenta circles, N = 37–180) unwinding activity. Maximum processivity at zero force determined from stopped-flow ensemble measurements for wtRep dimer (red open diamond) and RepΔ2B monomer (red filled diamond). The top axes show the forces applied to the hairpin (blue) or fork (magenta), and the bottom axis shows the corresponding destabilization energy. Error bars represent standard error of the median. The gray dashed lines in A–B, D indicate the extension expected for complete hairpin opening, placing a limit on the maximum attainable processivity. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Nucleic Acids Res, Volume 47, Issue 5, 23 January 2019, Pages 2523–2532, https://doi.org/10.1093/nar/gkz023 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Effect of duplex destabilization on unwinding speed of RepΔ2B and wtRep. (A–C) Representative sections of ... Figure 3. Effect of duplex destabilization on unwinding speed of RepΔ2B and wtRep. (A–C) Representative sections of hairpin DNA unwinding by wtRep dimer (A, shades of dark blue), hairpin DNA unwinding by RepΔ2B monomer (B, shades of blue), and fork DNA unwinding by RepΔ2B monomer (C, shades of magenta) showing unwinding speed with increasing destabilization energy. Color-coded dotted lines represent mean speed over all traces at the corresponding destabilization energy (see SI Text). The grey dashed line represents wtRep monomer (A) and RepΔ2B monomer (B, C) translocation speeds at zero force. For clarity, all selected traces start at the baseline (0 bp unwound) and extend to the first instance of a pause or a reversal in direction. (D) Mean unwinding speed as a function of destabilization energy for wtRep hairpin (open dark blue circles, N = 6–51), RepΔ2B hairpin (filled blue circles, N = 49–140), and RepΔ2B fork (filled magenta circles, N = 31–73) activity. The average unwinding speeds of wtRep dimer and RepΔ2B monomer at zero force are indicated by the red open and filled diamonds, respectively. The top axes show the forces applied to the hairpin (blue) or fork (magenta), and the bottom axis shows the corresponding destabilization energy. Error bars represent standard error of the mean. The grey dashed lines represent RepΔ2B and wtRep translocation speeds. The solid lines represent a model for the dependence of unwinding speed on destabilization energy for RepΔ2B (magenta) and wtRep (dark blue) (see main and SI text). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Nucleic Acids Res, Volume 47, Issue 5, 23 January 2019, Pages 2523–2532, https://doi.org/10.1093/nar/gkz023 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Helicase processivity is limited by strand-switching Figure 4. Helicase processivity is limited by strand-switching. (A, B) Representative trace of wtRep dimer (A) and ... Figure 4. Helicase processivity is limited by strand-switching. (A, B) Representative trace of wtRep dimer (A) and RepΔ2B monomer (B) activity on a hairpin DNA showing multiple rounds of unwinding and rezipping mediated by strand-switching (S). Close-up (insets) with individual data points (filled circles) shows that rezipping and unwinding have similar rates. The color-coded values in units of k<sub>B</sub>T correspond to the destabilization energies (see SI Text). (C) Representative trace of RepΔ2B monomer activity on fork DNA illustrating strand-switching (S), snap-back (B) and dissociation (D). Close-up (inset) shows that dissociation events are rapid and distinct from unwinding or rezipping (compare to insets in A, B). (D) Median strand-switching distance as a function of destabilization energy for wtRep hairpin (open dark blue circles, N = 50–120), RepΔ2B hairpin (filled blue circles, N = 11–66), and RepΔ2B fork (filled magenta circles, N = 12–44) activity. The top axes show the forces applied to the hairpin (blue) or fork (magenta), and the bottom axis shows the corresponding destabilization energy (see SI Text). The grey dashed lines in A–B, D indicate the limit set by the length of the DNA hairpin. The solid lines represent a model for the dependence of strand-switching distance on destabilization energy for RepΔ2B (magenta) and wtRep (dark blue) (see main and SI text). Error bars represent standard error of the median. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Nucleic Acids Res, Volume 47, Issue 5, 23 January 2019, Pages 2523–2532, https://doi.org/10.1093/nar/gkz023 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Schematic and model of RepΔ2B behaviors during DNA unwinding Figure 5. Schematic and model of RepΔ2B behaviors during DNA unwinding. Distinct protein behaviors, i.e. unwinding, ... Figure 5. Schematic and model of RepΔ2B behaviors during DNA unwinding. Distinct protein behaviors, i.e. unwinding, rezipping, strand-switching, dissociation, and looping (as labelled) are observed during wtRep dimer and RepΔ2B monomer activity in the optical trap measurements. Percentages correspond to the frequency of a behavior possible at each junction as indicated by the direction of the arrow. Percentage values represent averages over a 0.5-0.8 k<sub>B</sub>T destabilization energy range for wtRep hairpin (dark blue, N = 15), RepΔ2B hairpin (blue, N = 305) and RepΔ2B fork (magenta, N = 169) activity (see SI Text for details). The thickness of the arrow represents the frequency of a behavior at each junction. During strand-switching (schematics in dashed-line box), ssDNA dissociates from the canonical binding site (orange portion of the DNA), but non-canonical protein-DNA interactions (red circles) allow Rep to remain bound to the junction. The opposing strand binds to the canonical site to complete strand-switching. During snap-back (schematics in solid-line box), ssDNA dissociates from the canonical binding site. A loop formed via non-canonical binding (red circle) allows Rep to snap back to the 3′ end without dissociating. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Nucleic Acids Res, Volume 47, Issue 5, 23 January 2019, Pages 2523–2532, https://doi.org/10.1093/nar/gkz023 The content of this slide may be subject to copyright: please see the slide notes for details.