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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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Structures of gemifloxacin and levofloxacin, two antipneumococcal fluoroquinolones.
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gkt696-F1: Structures of gemifloxacin and levofloxacin, two antipneumococcal fluoroquinolones.

Mentions: A variety of clinically important antibacterial and anticancer drugs inhibit type II topoisomerases, sustaining interest in these enzymes as therapeutic targets (17,18). Anticancer agents such as etoposide target eukaryotic topo II, whereas antibacterial fluoroquinolones, e.g. gemifloxacin and levofloxacin, target topo IV and/or gyrase (Figure 1). The drugs interfere with DNA religation to stabilize a topoisomerase cleavage complex, which cellular processes convert into a cytotoxic lesion, possibly a double-stranded DNA break (19). Drug-arrested complexes are readily detected in vivo and in vitro by denaturation with sodium dodecyl sulphate (SDS), releasing double-stranded DNA breaks at specific sites (20,21). Although anticancer drug-promoted DNA cleavage has been extensively studied for eukaryotic topo II (22), much less is known about fluoroquinolone-mediated breakage by bacterial type II topoisomerases.Figure 1.


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

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

Structures of gemifloxacin and levofloxacin, two antipneumococcal fluoroquinolones.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt696-F1: Structures of gemifloxacin and levofloxacin, two antipneumococcal fluoroquinolones.
Mentions: A variety of clinically important antibacterial and anticancer drugs inhibit type II topoisomerases, sustaining interest in these enzymes as therapeutic targets (17,18). Anticancer agents such as etoposide target eukaryotic topo II, whereas antibacterial fluoroquinolones, e.g. gemifloxacin and levofloxacin, target topo IV and/or gyrase (Figure 1). The drugs interfere with DNA religation to stabilize a topoisomerase cleavage complex, which cellular processes convert into a cytotoxic lesion, possibly a double-stranded DNA break (19). Drug-arrested complexes are readily detected in vivo and in vitro by denaturation with sodium dodecyl sulphate (SDS), releasing double-stranded DNA breaks at specific sites (20,21). Although anticancer drug-promoted DNA cleavage has been extensively studied for eukaryotic topo II (22), much less is known about fluoroquinolone-mediated breakage by bacterial type II topoisomerases.Figure 1.

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