Journal: Nature Communications

Article Title: The free energy landscape of retroviral integration

doi: 10.1038/s41467-019-12649-w

Figure Lengend Snippet: Topology and yield of strand transfer intermediates. a Reaction schematic of PFV strand transfer with crystal structures of nucleoprotein intermediates (PDB 3l2r, 3os0, 3os1). b AFM image of untreated supercoiled plasmids. c Relative occurrence of supercoiled (SC), open circular (OC), and linear (LIN) topologies in untreated plasmid samples ( n tot = 214; errors are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sqrt n /n_{tot}$$\end{document} n ∕ n t o t ). d AFM image of supercoiled plasmids incubated with CI (10 nM; 4 h, 37 °C). e Relative occurrence of different free DNA topologies and nucleoprotein complexes, in samples of supercoiled plasmid incubated with cleaved intermediate (10 nM; 4 h, 37 °C) ( n tot = 518; errors are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sqrt n /n_{tot}$$\end{document} n ∕ n t o t ). f AFM image of sample after incubation with CI and subsequent deproteination. g Relative occurrence of DNA topological forms in deproteinated samples of supercoiled plasmid incubated with CI ( n tot = 100; errors are \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sqrt n /n_{tot}$$\end{document} n ∕ n t o t ). The linearized fraction increases significantly after deproteination compared with the other two conditions ( p < 0.0001 in both cases). h Gel electrophoresis of untreated plasmids (first lane), and native (secondlane) and deproteinated (third lane) reaction products of supercoiled pBR322 and intasomes assembled on Atto532-labeled viral DNA mimetics (left: ethidium bromide stain; right: Atto532 dye). i Ratio of full-site product to total DNA as a function of incubation time, deduced from electrophoretic separation of reaction mixtures of plasmids with different supercoiling density and CI intasome (25 nM), followed by Sybr Gold staining. Error bars are SD from two independent repeats. Source data are provided as a file

Article Snippet: Negatively supercoiled circular DNA plasmids (pUC19: 2686 bp, pBR322: 4361 bp, M13mp18 RF I DNA: 7249 bp) were obtained commercially (New England Biolabs, Ipswich, MA, USA).

Techniques: Plasmid Preparation, Incubation, Nucleic Acid Electrophoresis, Labeling, Staining

Journal: Nature Communications

Article Title: The free energy landscape of retroviral integration

doi: 10.1038/s41467-019-12649-w

Figure Lengend Snippet: Auxiliary DNA-binding interfaces are engaged in TCCs and STCs. a AFM images of CI intasomes. b Atomistic model of PFV intasome solution structure ( 21 ). Purple spheres indicate residues K168. Viral DNA mimetics (red) are not visible in our AFM data. c Early TCCs formed on linear pBR322 DNA reveal longitudinal and apical binding geometries. d Polar plots of entry and exit angles with respect to the intasome long axis for apical and longitudinal binding modes, and relative occurrence in WT and K168E intasomes. e AFM image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes. f AFM image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation. g AFM image of a gel-purified STC. h Polar plot of exit angles in branched complexes. i Polar plot of exit angles in bridging complexes. j Model for target DNA (blue) folding in TCC and STC intasomes (yellow dotted contour). Viral DNA mimetics are not shown. Source data are provided as a file

Article Snippet: Negatively supercoiled circular DNA plasmids (pUC19: 2686 bp, pBR322: 4361 bp, M13mp18 RF I DNA: 7249 bp) were obtained commercially (New England Biolabs, Ipswich, MA, USA).

Techniques: Binding Assay, Incubation, Plasmid Preparation, Purification

Journal: Nature Communications

Article Title: The free energy landscape of retroviral integration

doi: 10.1038/s41467-019-12649-w

Figure Lengend Snippet: Magnetic tweezers assay reveals target capture dynamics. a A typical field of view depicting ~50 beads used for tracking (yellow) and reference beads (blue). b DNA tethers are supercoiled using external magnets and exhibit extension fluctuations with SD σ z . Target capture reduces σ z via DNA bridging. Transient interface unbinding repositions intasomes thereby affecting extension. c Time trace of supercoiled target DNA extension before and after binding A188D CI intasomes (blue: raw data at 58 Hz, yellow: 1 Hz smoothed data). d Enlarged region of c highlighting the onset of dynamic bridging (green dotted line) and coinciding σ z reduction calculated with a 0.5 s moving window (red lines: trace fitted using step-finding algorithm) . e Exponential fit of Δt TCC A188D distribution yields a lifetime of auxiliary interfaces τ TCC A188D = 3.0 ± 0.5 s. (Error is 95% CI; n = 724). f Time trace of supercoiled DNA extension and response to binding WT CI in Ca 2+ buffer. g Enlarged region of f shows the onset of dynamic bridging and σ z reduction (green dotted line). h Exponential fit of Δt TCC Ca2+ distribution with lifetime τ TCC Ca2+ = 6.9 ± 1.3 s. (Error is 95% CI; n = 750). Source data are provided as a file

Article Snippet: Negatively supercoiled circular DNA plasmids (pUC19: 2686 bp, pBR322: 4361 bp, M13mp18 RF I DNA: 7249 bp) were obtained commercially (New England Biolabs, Ipswich, MA, USA).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: The free energy landscape of retroviral integration

doi: 10.1038/s41467-019-12649-w

Figure Lengend Snippet: Real-time observation of strand transfer and apical interface stability in STCs. a Scheme depicting signatures of strand transfer. Top: binding and reaction near plectoneme end loops minimally affect σ z but enables supercoil release through apical interface unbinding (green arrow) and rotational relaxation (black arrow). Bottom: binding near the origin of the plectoneme suppresses σ z , strand transfer quenches dynamic bridging by intasome-target anchoring. b , c Extension time-traces of supercoiled target DNA reacting with intasome, depicting stepwise extension increments ( b ) and extension hopping followed by a stable extension level ( c ). d Extension time-trace of supercoiled target DNA reacting with WT intasome. External magnets introduce supercoils that are released in steps due to transient apical interface unbinding. Extension plateaus quantify dwell times Δt Apic STC , step sizes Δz quantify extension increments. e In the plectonemic regime, the number of turns released per unbinding event ΔLk is proportional to Δz . f ΔLk distribution (kernel density estimate; bandwidth 0.2 turns). Inset: Fourier transformation after subtracting an exponential background. g Dependence of step size ΔLk distribution on the sign of ΔLk TCC in TCCs and on the sign of the torque Γ STC applied to STCs (red data points are mean < ΔLk > and error bars are 95% CI as obtained from an exponential fit; Supplementary Fig. ). h Dependence of dwell times Δt Apic STC on ΔLk TCC and Γ STC (red data points are mean lifetime τ Apic STC and error bars are 95% CI as obtained from an exponential fit; Supplementary Fig. ). Significance calculated using two-sample Kolmogorov–Smirnov test ( n TCC+ STC+ = 227; n TCC+ STC‒ = 288; n TCC− STC+ = 412 ; n TCC‒ STC‒ = 444). Source data are provided as a file

Article Snippet: Negatively supercoiled circular DNA plasmids (pUC19: 2686 bp, pBR322: 4361 bp, M13mp18 RF I DNA: 7249 bp) were obtained commercially (New England Biolabs, Ipswich, MA, USA).

Techniques: Binding Assay, Introduce, Transformation Assay

Journal: Nature Communications

Article Title: CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

doi: 10.1038/s41467-021-23535-9

Figure Lengend Snippet: a MpCAPP needs both NTPs and dNTPs for primer synthesis. In all, 4 µM MpCAPP wild-type protein was added into 10 ng/µl circular M13 ssDNA substrate in presence of 2.5 µM dNTPs (FAM-labelled dUTP) + 2.5–100 µM non-labelled NTPs and MpPrimBuffer. The reaction was incubated at 50 °C for 30 min and the products were resolved by denaturing PAGE. b MpCAPP FAM-labelled UTP incorporation is very poor. In total, 4 µM MpCAPP was added into 10 ng/µl circular M13 ssDNA substrate in presence of 2.5 µM NTPs (FAM-labelled UTP) + 2.5–100 µM non-labelled dNTPs and MpPrimBuffer. The reaction was incubated at 50 °C for 30 min. c MpCAPP priming is purine ribonucleotide dependent. In all, 1 µM MpCAPP was added into 1 µM mixed-sequence ssDNA substrate (oKZ53) in presence of 2.5 µM dNTPs (FAM-labelled dCTP) and 1 mM unlabelled NTPs as indicated in the figure. The reaction was incubated at 50 °C for 10 min. C – control reaction without protein, no – no NTPs, all – all NTPs, black arrow – signal of Cy5-labelled template. d DbCAPP primase activity is stimulated by addition of purine ribonucleotides. In total, 1 µM DpCAPP protein was added into 1 µM ssDNA (oKZ53) in presence of 2.5 µM dNTPs (FAM-labelled dCTP) and 1 mM unlabelled NTPs in DbPrimBuffer. The reaction was incubated at 50 °C for 10 min. Annotations identical as for panel c . nts – nucleotide length of DNA markers. Results shown are representative of three independent repeats (3a–d).

Article Snippet: MpCAPP primase assay: 20 μl reaction contained MpPrimBuffer (10 mM Bis-Tris Propane; pH 7, 0.5 mM TCEP, 10 mM MgCl 2 and 100 µM ZnCl 2 ), either 10 ng/µl circular M13 ssDNA (NEB) or 1 µM ssDNA substrate, MpCAPP proteins, unlabelled NTPs and dNTPs (NEB) and either FAM-labelled dUTP, FAM-labelled UTP, FAM-labelled dCTP or γ-phosphate Atto488-labelled GTP (NU-803-6FM, NU-821-6FM, NU-809-5FM, NU-834-488, Jena Bioscience) at combinations and concentrations indicated in the figure legends.

Techniques: Incubation, Sequencing, Activity Assay