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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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A. DNA bending and molecular interactions in the quinolone–E-site cleavage complex of S. pneumoniae topo IV. Top panel shows the 34-bp E-site DNA sequence highlighting the key positions engaged in topo IV-DNA and levofloxacin-DNA interactions. Bases in the −4 to +8 region are shown in bold. Active site ParC tyrosines that form a covalent 5′ phosphate link at +1 are shown in pink, intercalating quinolone–Mg2+ complexes (Q) and ParC isoleucine side chains are in dark green and red, respectively, and Mg2+ ions are shown in light green. Centre and bottom panels depict top and side views of the E-site DNA present in the levofloxacin–topo IV cleavage complex showing the disposition of drug molecules and DNA- and drug-chelated Mg2+ ions (centre) within the highly bent DNA. The smaller darker green sphere on each fluoroquinolone molecule corresponds to fluorine. Images were generated with First Glance in JMol based on protein data bank entry 3RAE.
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gkt696-F8: A. DNA bending and molecular interactions in the quinolone–E-site cleavage complex of S. pneumoniae topo IV. Top panel shows the 34-bp E-site DNA sequence highlighting the key positions engaged in topo IV-DNA and levofloxacin-DNA interactions. Bases in the −4 to +8 region are shown in bold. Active site ParC tyrosines that form a covalent 5′ phosphate link at +1 are shown in pink, intercalating quinolone–Mg2+ complexes (Q) and ParC isoleucine side chains are in dark green and red, respectively, and Mg2+ ions are shown in light green. Centre and bottom panels depict top and side views of the E-site DNA present in the levofloxacin–topo IV cleavage complex showing the disposition of drug molecules and DNA- and drug-chelated Mg2+ ions (centre) within the highly bent DNA. The smaller darker green sphere on each fluoroquinolone molecule corresponds to fluorine. Images were generated with First Glance in JMol based on protein data bank entry 3RAE.

Mentions: Analysis of Ca2+-promoted cleavage by topo IV allowed us to establish the determinants for intrinsic DNA recognition of a cleavage site by topo IV, namely G(N/a)A(N/a) *CACG (N/t)T(N/t)C (Figure 4 and Supplementary Figure S3). Comparison with gemifloxacin cleavage indicated that invariant base preferences at −4 to −1 on each strand are conferred primarily by the enzyme, whereas the drug contributes to specificity at +1/+4 and +2/+3 (Figures 4 and 5). Preferred bases map to key interactions in the quinolone-E-site cleavage complex of pneumococcal topo IV (27,28) wherein the gate DNA is sharply bent into a U-shape and cleaved with a drug molecule inserted between −1 and +1 bases on each strand (Figure 8). The bicyclic ring system of the quinolone (Figure 1) is stacked against the tyrosyl-linked +1 base, preventing reversal by distancing the reactive 5′ phosphotyrosine and 3′-OH DNA ends (Figure 8). Quinolones bind poly(dG) > poly(dA) > poly(dT) > poly(dC) (34) with the greater preferences for purines likely accruing through greater base stacking via π–π interactions with the aromatic system of the drug, explaining the +1 purine preference (Figures 3–5). The ParC I170 residues intercalate symmetrically from the minor groove between the +8 and +9 bases on each strand (Figure 8) (28) with the preferred −4G/+8C bases perhaps best accommodating side-chain intercalation to induce DNA bending. There is contact between the −3C of E-site DNA and ParC R28 but no clashes with A at the −3 position on the other strand accounting for the −3 base degeneracy. Surprisingly, no obvious enzyme contacts are seen with the highly preferred −2A/+6T bases (or at +2/+3 positions), but a ParE-chelated Mg2+ ion is bound to the non-scissile phosphodiester group linking −1 and −2 nucleotides on each strand, which might be sensitive to the bases present at these positions. Particular preferences at +2/+3 may allow establishment of the pre-cleavage B-A-B helix conformation at the gate (28) or facilitate adjustments needed for drug–DNA intercalation. Evidently, multiple enzyme, DNA, drug and metal ion interactions are involved in stabilizing the bent and cleaved DNA gate.Figure 8.


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

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

A. DNA bending and molecular interactions in the quinolone–E-site cleavage complex of S. pneumoniae topo IV. Top panel shows the 34-bp E-site DNA sequence highlighting the key positions engaged in topo IV-DNA and levofloxacin-DNA interactions. Bases in the −4 to +8 region are shown in bold. Active site ParC tyrosines that form a covalent 5′ phosphate link at +1 are shown in pink, intercalating quinolone–Mg2+ complexes (Q) and ParC isoleucine side chains are in dark green and red, respectively, and Mg2+ ions are shown in light green. Centre and bottom panels depict top and side views of the E-site DNA present in the levofloxacin–topo IV cleavage complex showing the disposition of drug molecules and DNA- and drug-chelated Mg2+ ions (centre) within the highly bent DNA. The smaller darker green sphere on each fluoroquinolone molecule corresponds to fluorine. Images were generated with First Glance in JMol based on protein data bank entry 3RAE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt696-F8: A. DNA bending and molecular interactions in the quinolone–E-site cleavage complex of S. pneumoniae topo IV. Top panel shows the 34-bp E-site DNA sequence highlighting the key positions engaged in topo IV-DNA and levofloxacin-DNA interactions. Bases in the −4 to +8 region are shown in bold. Active site ParC tyrosines that form a covalent 5′ phosphate link at +1 are shown in pink, intercalating quinolone–Mg2+ complexes (Q) and ParC isoleucine side chains are in dark green and red, respectively, and Mg2+ ions are shown in light green. Centre and bottom panels depict top and side views of the E-site DNA present in the levofloxacin–topo IV cleavage complex showing the disposition of drug molecules and DNA- and drug-chelated Mg2+ ions (centre) within the highly bent DNA. The smaller darker green sphere on each fluoroquinolone molecule corresponds to fluorine. Images were generated with First Glance in JMol based on protein data bank entry 3RAE.
Mentions: Analysis of Ca2+-promoted cleavage by topo IV allowed us to establish the determinants for intrinsic DNA recognition of a cleavage site by topo IV, namely G(N/a)A(N/a) *CACG (N/t)T(N/t)C (Figure 4 and Supplementary Figure S3). Comparison with gemifloxacin cleavage indicated that invariant base preferences at −4 to −1 on each strand are conferred primarily by the enzyme, whereas the drug contributes to specificity at +1/+4 and +2/+3 (Figures 4 and 5). Preferred bases map to key interactions in the quinolone-E-site cleavage complex of pneumococcal topo IV (27,28) wherein the gate DNA is sharply bent into a U-shape and cleaved with a drug molecule inserted between −1 and +1 bases on each strand (Figure 8). The bicyclic ring system of the quinolone (Figure 1) is stacked against the tyrosyl-linked +1 base, preventing reversal by distancing the reactive 5′ phosphotyrosine and 3′-OH DNA ends (Figure 8). Quinolones bind poly(dG) > poly(dA) > poly(dT) > poly(dC) (34) with the greater preferences for purines likely accruing through greater base stacking via π–π interactions with the aromatic system of the drug, explaining the +1 purine preference (Figures 3–5). The ParC I170 residues intercalate symmetrically from the minor groove between the +8 and +9 bases on each strand (Figure 8) (28) with the preferred −4G/+8C bases perhaps best accommodating side-chain intercalation to induce DNA bending. There is contact between the −3C of E-site DNA and ParC R28 but no clashes with A at the −3 position on the other strand accounting for the −3 base degeneracy. Surprisingly, no obvious enzyme contacts are seen with the highly preferred −2A/+6T bases (or at +2/+3 positions), but a ParE-chelated Mg2+ ion is bound to the non-scissile phosphodiester group linking −1 and −2 nucleotides on each strand, which might be sensitive to the bases present at these positions. Particular preferences at +2/+3 may allow establishment of the pre-cleavage B-A-B helix conformation at the gate (28) or facilitate adjustments needed for drug–DNA intercalation. Evidently, multiple enzyme, DNA, drug and metal ion interactions are involved in stabilizing the bent and cleaved DNA gate.Figure 8.

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