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Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases.

Arnoldi E, Pan XS, Fisher LM - Nucleic Acids Res. (2013)

Bottom Line: Analysis revealed strong enzyme-determined requirements for -4G, -2A and -1T bases preceding the breakage site (between -1 and +1) and enzyme-unique or degenerate determinants at -3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation.Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids.In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Sciences, St.George's, University of London, London SW17 0RE, UK.

ABSTRACT
Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca(2+)-induced cleavage distinguished 'intrinsic recognition' of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for -4G, -2A and -1T bases preceding the breakage site (between -1 and +1) and enzyme-unique or degenerate determinants at -3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.

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Analysis of DNA breakage at wt and mutant E-sites using a novel competitive cleavage assay. (A) Schematic diagram showing the basis of the assay. The wt and mutant (m1, m2 or m3) E-sites are amplified by PCR from plasmid pEA1 and its mutant variants to generate 276-, 256-, 236- and 216-bp fragments, respectively. Equal amounts of each of the four substrates are mixed and incubated with topo IV producing signature 176-, 156-, 136- and 116-bp cleavage fragments and a 100-bp product common to all sites. The four substrate and five cleavage fragments are separated by electrophoresis allowing the efficiency of cleavage at the mutant sites to be compared directly with wt. (B) A representative assay for gemifloxacin-promoted cleavage by topo IV. Panel (a), all four cleavage substrates (0.1 µg each) carried a wt E-site generating well-separated 176-, 156-, 136- and 116-bp products. Panels (b–h) each used a mix of wt E-site substrate (276-bp) with three mutant site substrates (256-, 236- and 216-bp) carrying changes at −4/+8 (panel (b)), −2/+6 (c), −1/+5 (d), −3/+7 (e and f), +1/+4 (g) and +2/+3 (h). All reactions contained topo IV and 6 mM MgCl2 and were carried out in the absence (±) or presence (+) of 320 µM gemifloxacin. Substrates and products were separated by electrophoresis in 4–12% gradient polyacrylamide gels and DNA bands were stained and photographed. Cleavage at each mutant site compared with the internal E-site control is indicated by the relative amounts of the cleavage products. Reduced fluorescence of the smaller cleavage products relative to the wt 176-bp band is largely compensated by the relatively greater number of sites presented per microgram of the smaller substrates. (C) Quantitation of cleavage products from wt and mutant E-sites in the competitive assay. Dark grey bar denotes the wt product. Data represent the average of two independent experiments: bars are standard error of the mean.
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gkt696-F3: Analysis of DNA breakage at wt and mutant E-sites using a novel competitive cleavage assay. (A) Schematic diagram showing the basis of the assay. The wt and mutant (m1, m2 or m3) E-sites are amplified by PCR from plasmid pEA1 and its mutant variants to generate 276-, 256-, 236- and 216-bp fragments, respectively. Equal amounts of each of the four substrates are mixed and incubated with topo IV producing signature 176-, 156-, 136- and 116-bp cleavage fragments and a 100-bp product common to all sites. The four substrate and five cleavage fragments are separated by electrophoresis allowing the efficiency of cleavage at the mutant sites to be compared directly with wt. (B) A representative assay for gemifloxacin-promoted cleavage by topo IV. Panel (a), all four cleavage substrates (0.1 µg each) carried a wt E-site generating well-separated 176-, 156-, 136- and 116-bp products. Panels (b–h) each used a mix of wt E-site substrate (276-bp) with three mutant site substrates (256-, 236- and 216-bp) carrying changes at −4/+8 (panel (b)), −2/+6 (c), −1/+5 (d), −3/+7 (e and f), +1/+4 (g) and +2/+3 (h). All reactions contained topo IV and 6 mM MgCl2 and were carried out in the absence (±) or presence (+) of 320 µM gemifloxacin. Substrates and products were separated by electrophoresis in 4–12% gradient polyacrylamide gels and DNA bands were stained and photographed. Cleavage at each mutant site compared with the internal E-site control is indicated by the relative amounts of the cleavage products. Reduced fluorescence of the smaller cleavage products relative to the wt 176-bp band is largely compensated by the relatively greater number of sites presented per microgram of the smaller substrates. (C) Quantitation of cleavage products from wt and mutant E-sites in the competitive assay. Dark grey bar denotes the wt product. Data represent the average of two independent experiments: bars are standard error of the mean.

Mentions: To investigate the biochemical requirements for DNA cleavage by bacterial type II topoisomerases, we used the E-site present in the S. pneumoniae parE gene (23). It is the strongest topo IV site within an 8-kb region of the pneumococcal chromosome and displays a high degree of 2-fold symmetry with −4G, −2A and −1T present on each strand corresponding to +8C, +6T and +5A on the complementary strand (Figure 2A). The E-site was amplified as a 256-bp PCR fragment and cloned into vector pCR2.1 TOPO to yield plasmid pEA1 (Figure 2B), which was used as a template in oligonucleotide-directed mutagenesis to introduce a variety of symmetric pairwise and single mutations into the −4 to +8 region (Figure 2C). The mutant plasmids were used to develop a new competitive assay in which wt and mutant E-sites (m1, m2 and m3) contained, respectively, on 276-, 256-, 236- and 216-bp PCR products (amplified from pEA1 and its mutants), are mixed in equal weight, cleaved in parallel by topo IV in the presence of fluoroquinolone and the signature 176-, 156-, 136- and 116-bp cleavage products (plus the 100-bp partner fragment) are separated and detected by gradient polyacrylamide gel electrophoresis (Figure 3A). By providing a choice of independent substrates, the assay compares breakage at mutant sites with that of the wt site internal control and requires only a few gel lanes to display the full cleavage repertoire.Figure 2.


Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases.

Arnoldi E, Pan XS, Fisher LM - Nucleic Acids Res. (2013)

Analysis of DNA breakage at wt and mutant E-sites using a novel competitive cleavage assay. (A) Schematic diagram showing the basis of the assay. The wt and mutant (m1, m2 or m3) E-sites are amplified by PCR from plasmid pEA1 and its mutant variants to generate 276-, 256-, 236- and 216-bp fragments, respectively. Equal amounts of each of the four substrates are mixed and incubated with topo IV producing signature 176-, 156-, 136- and 116-bp cleavage fragments and a 100-bp product common to all sites. The four substrate and five cleavage fragments are separated by electrophoresis allowing the efficiency of cleavage at the mutant sites to be compared directly with wt. (B) A representative assay for gemifloxacin-promoted cleavage by topo IV. Panel (a), all four cleavage substrates (0.1 µg each) carried a wt E-site generating well-separated 176-, 156-, 136- and 116-bp products. Panels (b–h) each used a mix of wt E-site substrate (276-bp) with three mutant site substrates (256-, 236- and 216-bp) carrying changes at −4/+8 (panel (b)), −2/+6 (c), −1/+5 (d), −3/+7 (e and f), +1/+4 (g) and +2/+3 (h). All reactions contained topo IV and 6 mM MgCl2 and were carried out in the absence (±) or presence (+) of 320 µM gemifloxacin. Substrates and products were separated by electrophoresis in 4–12% gradient polyacrylamide gels and DNA bands were stained and photographed. Cleavage at each mutant site compared with the internal E-site control is indicated by the relative amounts of the cleavage products. Reduced fluorescence of the smaller cleavage products relative to the wt 176-bp band is largely compensated by the relatively greater number of sites presented per microgram of the smaller substrates. (C) Quantitation of cleavage products from wt and mutant E-sites in the competitive assay. Dark grey bar denotes the wt product. Data represent the average of two independent experiments: bars are standard error of the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814380&req=5

gkt696-F3: Analysis of DNA breakage at wt and mutant E-sites using a novel competitive cleavage assay. (A) Schematic diagram showing the basis of the assay. The wt and mutant (m1, m2 or m3) E-sites are amplified by PCR from plasmid pEA1 and its mutant variants to generate 276-, 256-, 236- and 216-bp fragments, respectively. Equal amounts of each of the four substrates are mixed and incubated with topo IV producing signature 176-, 156-, 136- and 116-bp cleavage fragments and a 100-bp product common to all sites. The four substrate and five cleavage fragments are separated by electrophoresis allowing the efficiency of cleavage at the mutant sites to be compared directly with wt. (B) A representative assay for gemifloxacin-promoted cleavage by topo IV. Panel (a), all four cleavage substrates (0.1 µg each) carried a wt E-site generating well-separated 176-, 156-, 136- and 116-bp products. Panels (b–h) each used a mix of wt E-site substrate (276-bp) with three mutant site substrates (256-, 236- and 216-bp) carrying changes at −4/+8 (panel (b)), −2/+6 (c), −1/+5 (d), −3/+7 (e and f), +1/+4 (g) and +2/+3 (h). All reactions contained topo IV and 6 mM MgCl2 and were carried out in the absence (±) or presence (+) of 320 µM gemifloxacin. Substrates and products were separated by electrophoresis in 4–12% gradient polyacrylamide gels and DNA bands were stained and photographed. Cleavage at each mutant site compared with the internal E-site control is indicated by the relative amounts of the cleavage products. Reduced fluorescence of the smaller cleavage products relative to the wt 176-bp band is largely compensated by the relatively greater number of sites presented per microgram of the smaller substrates. (C) Quantitation of cleavage products from wt and mutant E-sites in the competitive assay. Dark grey bar denotes the wt product. Data represent the average of two independent experiments: bars are standard error of the mean.
Mentions: To investigate the biochemical requirements for DNA cleavage by bacterial type II topoisomerases, we used the E-site present in the S. pneumoniae parE gene (23). It is the strongest topo IV site within an 8-kb region of the pneumococcal chromosome and displays a high degree of 2-fold symmetry with −4G, −2A and −1T present on each strand corresponding to +8C, +6T and +5A on the complementary strand (Figure 2A). The E-site was amplified as a 256-bp PCR fragment and cloned into vector pCR2.1 TOPO to yield plasmid pEA1 (Figure 2B), which was used as a template in oligonucleotide-directed mutagenesis to introduce a variety of symmetric pairwise and single mutations into the −4 to +8 region (Figure 2C). The mutant plasmids were used to develop a new competitive assay in which wt and mutant E-sites (m1, m2 and m3) contained, respectively, on 276-, 256-, 236- and 216-bp PCR products (amplified from pEA1 and its mutants), are mixed in equal weight, cleaved in parallel by topo IV in the presence of fluoroquinolone and the signature 176-, 156-, 136- and 116-bp cleavage products (plus the 100-bp partner fragment) are separated and detected by gradient polyacrylamide gel electrophoresis (Figure 3A). By providing a choice of independent substrates, the assay compares breakage at mutant sites with that of the wt site internal control and requires only a few gel lanes to display the full cleavage repertoire.Figure 2.

Bottom Line: Analysis revealed strong enzyme-determined requirements for -4G, -2A and -1T bases preceding the breakage site (between -1 and +1) and enzyme-unique or degenerate determinants at -3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation.Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids.In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Sciences, St.George's, University of London, London SW17 0RE, UK.

ABSTRACT
Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca(2+)-induced cleavage distinguished 'intrinsic recognition' of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for -4G, -2A and -1T bases preceding the breakage site (between -1 and +1) and enzyme-unique or degenerate determinants at -3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.

Show MeSH
Related in: MedlinePlus