<i>In vitro</i> characterisation of DNA gyrase screen hits.

<p>A. Determination of the IC₅₀ for mitoxantrone in a supercoiling assay with 1 unit of gyrase (12 nM); 100 μM ciprofloxacin (Cip.) was used as a positive control for inhibition. The positions of relaxed (Rel.) and negatively supercoiled (SC) DNA are indicated. B. Determination of the IC₅₀ for suramin. C. Assaying the abilities of mitoxantrone and suramin to induce gyrase-mediated DNA cleavage. The reactions were carried out in the absence of ATP. Ciprofloxacin was used as a positive control for gyrase-mediated cleavage. The position of linear DNA (Lin.) is indicated. D. Suramin-induced protection of DNA from Ca²⁺-induced, gyrase-mediated cleavage. E. Inhibition of gyrase binding to a 147 bp DNA fragment by suramin.</p

<i>In vitro</i> characterisation of topoisomerase VI screen hits.

<p>A. Determination of IC₅₀ values for hits in the presence of 1 unit (50 nM) of <i>M. mazei</i> topo VI. This resulted in the following IC₅₀ values: 6 μM for 9-aminoacridne; 30 μM for m-amsacrine; 30 μM for suramin; 30 μM for hexylresorcinol; 40 μM for purpurin; 8 μM for quinacrine; and 2 μM for mitoxanthrone. B. Native gel shift assays for the binding of <i>M. mazei</i> topo VI to a 147 bp DNA fragment in the presence of screen hits.</p

Summary of Screen Hits.

*<p>Not determined in this study.</p

DNA cleavage assays with topoisomerase VI screen hits.

<p>A. Assaying the abilities of screen hits to induce <i>M. mazei</i> topo VI-mediated DNA cleavage with 1 unit topo VI (50 nM). B. Inhibition of <i>S. shibatae</i> topo VI by screen hits. C. Assaying the abilities of screen hits to induce <i>S. shibatae</i> topo VI-mediated DNA cleavage. D. Protection of DNA from ADPNP-induced, <i>S. shibatae</i> topo VI-mediated cleavage by screen hits.</p

Inhibition of <i>Arabidopsis</i> hypocotyl extension by hexylresorcinol.

<p>All plants were grown for 5 days in the dark. A. Average length of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol. Error bars represent the standard deviation of the samples. B. Percentage germination of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol.</p

Growth of <i>M. smegmatis</i> mc²155 in the presence of mitoxantrone or suramin.

<p>A. Bacteria were grown in liquid cultures in the presence of drug for 22 hours, and then plated onto drug-free agar. After a further 48 h the colonies were counted for each concentration of drug. B. Colony counts for bacteria grown in the presence of 0, 13 or 65 µM mitoxantrone for 16, 19 or 22 hours before being plated onto solid media.</p

DNA gyrase-mediated cleavage of DNA, stimulated by CcdB.

<p>A. Negatively supercoiled pBR322 (3.5 nM) was incubated with 30 nM gyrase, 1.4 mM ATP and various concentrations of CcdB between 0.001–10 µM, as indicated, for 1 h at 25°C. Cleaved DNA was revealed by the addition of SDS and proteinase K. After incubation for 30 mins at 37°C, reactions were stopped with STEB and DNA was subjected to phenol extraction, and samples were analysed on a 1% agarose gel. N, nicked DNA; L, linear DNA; SC, supercoiled DNA. (B) CcdA inhibits the action of CcdB. Negatively supercoiled pBR322 was incubated with gyrase as described in (A) in the presence of 1.4 mM ATP, with 1 µM CcdB and various concentrations of CcdA between 0.1–10 µM as indicated. N, nicked DNA; L, linear DNA; SC, supercoiled DNA.</p

NMS profile of the interactions of Kid with CcdA proteins at different toxin-antitoxin ratios.

<p>(A), Mass spectrum obtained at a Kid∶CcdA ratio of 2∶1. (B), Mass spectrum obtained at a Kid∶CcdA ratio of 1∶1.(C) Mass spectrum obtained at a Kid∶CcdA ratio of 1∶2. All the protein mixtures were performed in 100 mM ammonium acetate pH 6.7. The lowest protein concentration was 10 µM in all cases. CcdA protein is represented with maroon circles and Kid protein is represented with blue squares. Each complex found is represented with the appropriate combination of squares and circles.</p

NMS profile of the interactions of CcdB and His₆Kis proteins at different toxin-antitoxin ratios.

<p>(A) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 2∶1. (B) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 1∶1. (C) Mass spectrum otained at a CcdB∶His₆Kis molar ratio of 1∶2. (D) Mass spectrum obtained at a CcdB∶His₆Kis molar ratio of 1∶4. All the protein mixtures were performed in 100 mM ammonium acetate pH 6.7. The lowest protein concentration was 10 µM in all cases except the 1∶4 ratio that was 5 µM. CcdB protein is represented with green squares and His₆Kis with yellow circles. Each complex found is represented with the appropriate combination of squares and circles.</p

<i>In vitro</i> analysis of RNA cleavage by Kid/Kis and CcdB/CcdA.

<p>(A) 5′ ³²P-labelled CopT RNA was incubated for 2 min at 37°C in the presence of Kid or CcdB prior to separation on 8% polyacrylamide gels in the presence of urea. Tracks: 1, C−, untreated full-length (FL) RNA; 2, RNA treated with 0.2 µM Kid; 3, RNA treated with 0.2 µM Kid and 0.2 µM Kis; 4, RNA treated with 0.2 µM Kid and 0.2 µM CcdA; 5, RNA treated with 0.2 µM CcdA; 6, RNA treated with 0.2 µM CcdB; 7, RNA treated with 0.2 µM CcdA and 0.2 µM CcdB; 8, RNA treated with 0.2 µM Kid and 1.5 µM BSA; 9, RNA treated with 0.2 µM Kid and 0.2 µM CcdB. (B) Bar graph representation and quantitative analysis of data in (A): Total RNA (uncleaved and cleaved products) was calculated for each track scanning the different bands using the Quantity One® program (Bio-Rad). RNase activity was calculated as the percentage of full length RNA substrate. The average value for each condition was calculated from three independent experiments. The 100% value indicates absence of RNase activity. The standard deviation is indicated above the different bars. A Student's t-test indicated that the differences between values in lanes 2 and 3, linked with a bracket, were significant (p-value = 0.0067). ** Represents a p-value≤0.01.</p