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: eLife

Article Title: A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

doi: 10.7554/elife.45393

Figure Lengend Snippet: Figure 5. RNase J2 promotes RNase and DNase activities of RNase J1. (A) Purified recombinant ribonucleases J1, J2, and a catalytically-dead variant of J1 bearing H74A and H76A mutations (dJ1). Proteins were resolved by SDS- PAGE and visualized using Coomassie G-250 staining. (B) RNA and DNA substrates used in nuclease assays. (C) Ribonuclease activities of RNases J1 and J2. The 5’-end labelled 31-nucleotide RNA substrate was combined with 0.5 pmols of indicated enzyme(s). The reaction mixture was incubated at 37˚C for increasing time points (0, 5, 10, or 20 min.) RNAs were resolved using denaturing urea-PAGE. (D) Quantification of ribonuclease activity. Shown is the average fraction RNA cleaved (±S.D.) of three independent trials (see Figure 5—source data 1). (E) Deoxyribonuclease activities of RNases J1 and J2 on linear single-stranded DNA. The 5’-end labelled 60- nucleotide DNA substrate was combined with 0.5 pmols of indicated enzyme(s). The reaction mixture was incubated at 37˚C for increasing time points (0, 2, 5, or 10 min.) DNAs were resolved using denaturing urea-PAGE. (F) Quantification of deoxyribonuclease activity on single-stranded DNA. The average fraction DNA cleaved (±S.D.) from three independent trials is shown (see Figure 5—source data 2). (G) Deoxyribonuclease activities of RNases J1 and J2 on double-stranded DNA substrates. The indicated enzymes (two pmols) were combined with 0.1 mg pUC19 DNA that was supercoiled or linearized by PstI digestion. The reaction mixture was incubated at 37˚C for 1 hr and DNA was resolved on an agarose gel. (H) Deoxyribonuclease activities of RNases J1 and J2 on single- stranded circular DNA. The indicated enzyme(s) (four pmols) were combined with 0.5 mg of phage M13MP18 DNA and the reaction mixture was incubated at 37˚C for increasing time points (15, 30, 60 min.) The DNA was resolved on a 0.7% agarose gel. Images in panels G and H are representatives of four independent trials. DOI: https://doi.org/10.7554/eLife.45393.017 The following source data and figure supplement are available for figure 5:

Article Snippet: DOI: https://doi.org/10.7554/eLife.45393 16 of 25 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pET28b-His10Smt3-drnjA This paper for RNase J1/H74A H76A overexpression and purification, see Supplementary file 1 Recombinant DNA reagent pUC19 New England Biolabs Cat. # N3041S Recombinant DNA reagent M13MP18 New England Biolabs Cat. # N4040S Sequencebased reagent DNA oligonucleotides (multiple) Eurofins MWG Operon To build and sequence recombinant DNA constructs, and to perform quantitative PCR, see Supplementary file 1 Peptide, recombinant protein T4 Polynucleotide kinase New England Biolabs Cat. # M0201L Peptide, recombinant protein Lysostaphin AmbiProducts via Fisher Cat. # NC0318863 Peptide, recombinant protein DNase I New England Biolabs Cat. # M0303S Peptide, recombinant protein M-MuLV reverse transcriptase New England Biolabs Cat. # M0253L Peptide, recombinant protein DpnI New England Biolabs Cat. # R0176S Peptide, recombinant protein SUMO Protease MCLAB, http://www.mclab.com /SUMO-Protease.html Cat. # SP-100 Commercial assay or kit EZNA Cycle Pure Kit Omega Biotek via VWR Cat. # 101318–892 Commercial assay or kit EZNA Plasmid DNA Mini Kit Omega Biotek via VWR Cat. # 101318–898 Commercial assay or kit Wizard Genomic DNA Purification Kit Promega Corporation via VWR Cat. # A1120 Commercial assay or kit PerfeCTa SYBR Green SuperMix Quanta Biosciences via VWR Cat. # 101414–150 Chemical compound, drug TRIzol Reagent Thermo Fisher Cat. # 15596026 Chemical compound, drug TRI REAGENT RT Fisher Scientific Cat. # NC0850610 Chemical compound, drug BAN (4-bromoanisole) Fisher Scientific Cat. # NC9734505 Chemical compound, drug g-32P-ATP Perkin Elmer Cat. # BLU502H250UC Chemical compound, drug HisPur Ni-NTA Resin Thermo Fisher Cat. # 88222 Software, algorithm ImageQuant TL GE Healthcare/ Life Sciences RRID: SCR_014246 Version 8.1, used for densitometry Continued on next page Chou-Zheng and Hatoum-Aslan. eLife 2019;8:e45393.

Techniques: Purification, Recombinant, Variant Assay, SDS Page, Staining, Incubation, Activity Assay, Agarose Gel Electrophoresis