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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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Drug structure-specific preferences at +1. Symmetric +1/+4 mutant E-sites were mixed with the wt site (+1G/+4T), cleaved with topo IV in the presence of gemifloxacin and levofloxacin at the concentrations shown, and the products were separated and analysed on 4–12% polyacrylamide gradient TBE gels as described in the Figure 3 legend. A 100-bp cleavage product is generated by breakage at any cleavable E-site.
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gkt696-F5: Drug structure-specific preferences at +1. Symmetric +1/+4 mutant E-sites were mixed with the wt site (+1G/+4T), cleaved with topo IV in the presence of gemifloxacin and levofloxacin at the concentrations shown, and the products were separated and analysed on 4–12% polyacrylamide gradient TBE gels as described in the Figure 3 legend. A 100-bp cleavage product is generated by breakage at any cleavable E-site.

Mentions: The drug-mediated preference for purines at +1 positions (Figure 3 and 4) is interesting given that recent X-ray crystal structures of the topo IV–E-site cleavage complex reveal two quinolone molecules stacked against the +1 bases (27,28). To examine the sequence preferences of different quinolones, we compared topo IV cleavage of linear pEA1 plasmid mediated by six fluoroquinolones currently in clinical use: the antipneumococcal drugs gemifloxacin, trovafloxacin, moxifloxacin, levofloxacin and sparfloxacin, plus ciprofloxacin used against Gram-negative pathogens (Supplementary Figure S4). All six quinolones promoted cleavage at a similar spectrum of sites including the E-site but with efficiencies determined by the drug. We selected the structurally distinct gemifloxacin and levofloxacin (Figure 1) for more detailed comparison using the competitive assay and the −4, −2 and +1 E-site mutants used in Figure 3B. Levofloxacin is less potent than gemifloxacin (32) and therefore a range of different drug concentrations was used to facilitate comparison at similar levels of cleavage. Levofloxacin recapitulated the −4 and −2 preferences seen in Figure 3 for gemifloxacin (results not shown). However, whereas gemifloxacin was able to promote cleavage of E-sites with a variety of purine/pyrimidine combinations at +1/+4, levofloxacin showed a marked preference for the +1G/+4C substrate yielding predominantly the G/C cleavage product rather than the wt (G/T) product and with little or no breakage of C/G and A/T sites (Figure 5). These biochemical results show directly for the first time that cleavage preferences at the +1 positions are modulated by the molecular structure of the quinolone.Figure 5.


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

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

Drug structure-specific preferences at +1. Symmetric +1/+4 mutant E-sites were mixed with the wt site (+1G/+4T), cleaved with topo IV in the presence of gemifloxacin and levofloxacin at the concentrations shown, and the products were separated and analysed on 4–12% polyacrylamide gradient TBE gels as described in the Figure 3 legend. A 100-bp cleavage product is generated by breakage at any cleavable E-site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt696-F5: Drug structure-specific preferences at +1. Symmetric +1/+4 mutant E-sites were mixed with the wt site (+1G/+4T), cleaved with topo IV in the presence of gemifloxacin and levofloxacin at the concentrations shown, and the products were separated and analysed on 4–12% polyacrylamide gradient TBE gels as described in the Figure 3 legend. A 100-bp cleavage product is generated by breakage at any cleavable E-site.
Mentions: The drug-mediated preference for purines at +1 positions (Figure 3 and 4) is interesting given that recent X-ray crystal structures of the topo IV–E-site cleavage complex reveal two quinolone molecules stacked against the +1 bases (27,28). To examine the sequence preferences of different quinolones, we compared topo IV cleavage of linear pEA1 plasmid mediated by six fluoroquinolones currently in clinical use: the antipneumococcal drugs gemifloxacin, trovafloxacin, moxifloxacin, levofloxacin and sparfloxacin, plus ciprofloxacin used against Gram-negative pathogens (Supplementary Figure S4). All six quinolones promoted cleavage at a similar spectrum of sites including the E-site but with efficiencies determined by the drug. We selected the structurally distinct gemifloxacin and levofloxacin (Figure 1) for more detailed comparison using the competitive assay and the −4, −2 and +1 E-site mutants used in Figure 3B. Levofloxacin is less potent than gemifloxacin (32) and therefore a range of different drug concentrations was used to facilitate comparison at similar levels of cleavage. Levofloxacin recapitulated the −4 and −2 preferences seen in Figure 3 for gemifloxacin (results not shown). However, whereas gemifloxacin was able to promote cleavage of E-sites with a variety of purine/pyrimidine combinations at +1/+4, levofloxacin showed a marked preference for the +1G/+4C substrate yielding predominantly the G/C cleavage product rather than the wt (G/T) product and with little or no breakage of C/G and A/T sites (Figure 5). These biochemical results show directly for the first time that cleavage preferences at the +1 positions are modulated by the molecular structure of the quinolone.Figure 5.

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