IN specific and aspecific binding to HIV DNA mimetics.

a. Schematic representation of the experimental set up for AFM imaging experiments. b. Schematic of the 491 bp DNA used to mimic the HIV cDNA, both as a blunt ended (BE) or as a 3’-preprocessed substrate (PE). c. Overview AFM topograph of the 491 bp DNA in the presence of 100 nM IN. Color range is 1.5 nm. d. HIV DNA mimetic with end-bound IN. e. HIV DNA mimetic with integrase bound at internal sites and definition of bend angle (inset). f. HIV DNA mimetic with integrase-mediated DNA looping. g. Selectivity for end-binding over binding to internal sites in the mimetic as a function of 3’ processing and buffer conditions. From left to right: blunt DNA ends, in the presence of Ca2+; blunt DNA ends, in the presence of Mg2+; processed DNA ends, in the presence of Mg2+. The IN binding site size is assumed to be 16 bp. Error bars are based on counting statistics. h. Bend angle distribution measured at 10 nm length scale for bare DNA and IN-DNA nucleoprotein complexes (NPCs) deposited from near-physiological salt buffer (50 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl pH = 7.4; N = 232) or high salt buffer (250 mM NaCl, 5 mM MgCl2, 10 mM Tris-HCl pH = 7.4; N = 109). i. Histogram of the normalized volume of IN free in solution (red) and IN bound to DNA (blue). In free solution, we find a small fraction of IN monomers and a dominant fraction of IN dimers (inset). For IN bound to DNA, we find the resulting protein volume distribution to be significantly broader and shifted to larger volumes as compared to the DNA-unbound IN population.

IN compacts long viral DNA mimetics into biphasic condensates.

a. AFM topographs of 4.8 kbp DNA in the presence of (from left to right) increasing IN concentrations. b. Radius of gyration of the nucleoprotein complex for 4.8 kbp DNA at 60 mM ionic strength as a function of [IN]. The data reveal two distinct compaction transition and are fitted by a double-Hill-fit: the first transition from open state to rosette state occurs at ∼ 500 nM, the second transition from rosette state to fully compacted state at ∼ 1100 nM. Representative AFM images are shown as insets on the right. Symbols and error bars are the mean and s.e.m. from typically ≥ 100 molecules per condition. c. Phase diagram depicting the normalized radius of gyration of the nucleoprotein complexes formed as a function of [IN] and DNA length, normalized to the largest Rg observed for each DNA length. With increasing DNA length, the compaction transitions shift to lower IN concentration. Intriguingly, at DNA length > 3.4 kbp a biphasic compaction transition is observed, whereas at 3.4 kbp the transition occurs in one step.

Monte Carlo (MC) simulations show that IN protein-protein interactions are required for biphasic compaction.

a. Schematic representation of the setup for MC simulations. DNA beads (4 nm) are colored in light blue and IN beads (same size) are colored in yellow. The box size is not to scale. b. Principal component analysis separates the large dataset of DNA-IN complexes into several categories of DNA-IN complexes (depicted as different colors, which were assigned using a Gaussian mixture model). The grey trajectories indicate two typical simulation paths of 4.8 kbp DNA (408 beads). Without IN-IN attraction, the left path is taken (triangles at start and end point), here no rosette intermediates are formed. Only if IN-IN attractions are added (right path; stars at start and end point), rosette formation (yellow) as well as full compaction are possible (red). c-g. Some typical conformations along the pathways in panel b. The scale bar indicates 40 nm. Free IN are not depicted. h. IN-DNA binding probability (mean ± SD from 5 independent simulations) as a function of binding strength (in kBT) from MC simulations. The horizontal lines indicate the experimentally determined binding probability for 100 nM and 200 nM IN from AFM images. The crossing area of experiment and simulations allows to estimate the IN-DNA binding energy to be 4.5-5 kBT. i. Time evolution of the state of compaction (same color as in panel b) of a 4.8 kbp DNA during a MC simulation with IN-IN attractions for varying IN concentrations, well in line with the experimental data (Fig. 2). Each simulation was selected as the most typical one from 12 simulations per IN concentration.

AFM force-volume based multiparametric imaging of IN-DNA condensates.

a. Schematic depiction of AFM-based force-volume mapping, and derived parameters k (indentation stiffness), Eplast. (plastic deformation energy), and Eelast. (elastic deformation energy). b. Example force distance curves (yellow: approach; black: retract). c. Multiparametric imaging of a condensate in the rosette state: zero-force height (top left), stiffness (top right), elastic energy (bottom left), plastic energy (bottom right). d. Multiparametric imaging of a DNA-IN condensate in the fully compacted state, indicating a soft “coat” surrounding a rigid “core”. e. Viscoelasticity index (mean ± SD) of the core and coat of fully compacted state, and of core of rosette state. p-values are based on 2-sample Kolmogorov-Smirnov test. f. Indentation stiffness (mean ± SD) of the core and coat of fully compacted state, and of core of rosette state. p-values are based on 2-sample Kolmogorov-Smirnov test.

Single-molecule force spectroscopy quantifies dynamics condensate unfolding.

a. Schematic depiction of the MT force spectroscopy measurements. 21 kbp DNA is tethered between a flow cell surface and magnetic beads and (partially) compacted by IN. External magnets enable the application of precisely calibrated stretching forces. b. Force-extension relation at forces ≤5 pN. Extension z is normalized to L0, the extension of bare DNA at F = 5 pN. Red line is a worm-like chain (WLC) fit of bare DNA. The inset shows the deviation of the experimental data (mean ± SD) in the presence of IN from bare DNA WLC behavior; the maximum deviation occurs at ∼ 0.3 pN. c. Extension-time traces of IN-DNA condensates at constant forces ≤0.3 pN exhibiting dynamic compaction and extension. d. Top: Extension-time trace following a force jump from 0.01 to 1 pN. Raw data (68 Hz) are depicted in black, yellow line is smoothed data (Butterworth filter, see Methods and SI for filter parameters). Insets depict a schematic of the events governing dwells (corresponding to extension plateaus), and extension bursts following dissociation of IN-DNA bridge in the core of the complex (red arrow). Bottom: Velocity-time trace, as obtained by differentiation of the filtered extension trace. Red marks indicate extension bursts detected by thresholding. e. Burst size distribution (blue bars; N= 777 events from 41 independent DNA tethers). For comparison, the loop size distribution obtained by AFM imaging are shown (red bars). A two sample t-test suggest no significant difference between both distributions (2-sample t-test; p = 0.5) f. Weighted bursts velocity distribution fitted with exponential decay (blue line; fitted decay constant 12 ± 3 nm/s) and power law (red line; scaling exponent –1.9 ± 0.3) for 1 nm/s < v < 100 nm/s. g. Distribution of dwell times between extension bursts and fits using an exponential decay (blue line; decay time of 24 ± 3 s) and power law (red line; scaling exponent –1.0 ± 0.1). Errors reflect 95% confidence intervals on the fit parameter.

Allosteric inhibitors affect compaction behavior of long viral DNA mimetics.

a. AFM topographs of the 4.8 kbp DNA in the presence of 1000 nM IN (rosette state) and no, 1000 nM, 2000 nM inhibitor. The inhibitor was added after complex formation. b. Rg-distributions for the entire molecule at 1000 nM IN and no, 1000 nM, 2000 nM inhibitors. As a reference, the distribution for bare DNA is shown as well. With increasing inhibitor concentration, less compaction is observed. c. Same conditions and color coding as in panel b only the Rg-distributions for the core are shown. d. AFM topographs of the 4.8 kbp DNA in the presence of 1500 nM IN (fully compacted state) and no, 1500 nM, 3000 nM inhibitor. The inhibitor was added after complex formation. e. Rg-distributions at 1500 nM IN and no, 1500 nM, 3000 nM inhibitors. As a reference, the distribution for bare DNA is shown as well. f. Same conditions and color coding as in panel e only the Rg-distributions for the core are shown. Even at high inhibitor concentrations, the fully compacted DNA-IN complexes do not disassemble under influence of the inhibitor. The insets in panels b, c, e, and f shows the means ± SEM of the respective distributions. Significance values are based on 2-sample Kolmogorov-Smirnov test: * indicates p < 0.05; *** indicate p < 0.001, n.s. indicates not significant.

Model for IN-mediated DNA compaction.

a. Schematic model of DNA compaction by IN. At increasing IN concentrations, the protein binds to the DNA and can form DNA loops. At intermediate concentration, loop-extruding complexes, so-called rosettes, form. At high IN concentrations, full compaction occurs. b. The allosteric IN inhibitor CX014442 can de-compact rosettes. c. The allosteric IN inhibitor CX014442 cannot de-compact fully compacted DNA-IN condensates. We observe that all compaction and de-compaction processes are reversible.