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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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Gate mutations at E- and V-sites inhibit topo IV cleavage. ScaI-linearized plasmid pEA1 bearing wt or mutant E- and V-sites was incubated with topo IV alone (lanes 1–4) or in the presence of 2.5 µM gemifloxacin (lanes 5–8) or 32 mM Ca2+ (lanes 9–12). DNA cleavage was induced as described in previous figure legends and DNA products were examined by electrophoresis in a 1% agarose gel. Cleavage fragments V1 and V2, and E1 and E2, arising from breakage at the V- and E-sites are indicated by arrows. The presence of wt or mutant sites is indicated above each lane: −4C and −2T denote the presence of pairwise −4C/+8G or −2T/+6T mutations, respectively. M, DNA size markers.
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gkt696-F6: Gate mutations at E- and V-sites inhibit topo IV cleavage. ScaI-linearized plasmid pEA1 bearing wt or mutant E- and V-sites was incubated with topo IV alone (lanes 1–4) or in the presence of 2.5 µM gemifloxacin (lanes 5–8) or 32 mM Ca2+ (lanes 9–12). DNA cleavage was induced as described in previous figure legends and DNA products were examined by electrophoresis in a 1% agarose gel. Cleavage fragments V1 and V2, and E1 and E2, arising from breakage at the V- and E-sites are indicated by arrows. The presence of wt or mutant sites is indicated above each lane: −4C and −2T denote the presence of pairwise −4C/+8G or −2T/+6T mutations, respectively. M, DNA size markers.

Mentions: To compare topo IV action at E- and V-sites, we used linear pEA1 plasmid substrates bearing mutations at one or both sites in the presence of either 6 mM MgCl2 and gemifloxacin, or 32 mM Ca2+ ions (Figure 6). In the absence of drug or Ca2+, there was no topo IV-induced plasmid breakage (lanes 1–4). Inclusion of gemifloxacin promoted cleavage at a spectrum of sites, and as expected substitution of guanines at −4 with disfavoured cytosines abrogated gemifloxacin-stimulated cleavage at the E-site eliminating the E1 and E2 products (Figure 6, cf lanes 5 and 6). Introduction of −4C mutations or −2T changes on both strands of the V-site blocked cleavage at the site indicated by the absence of V1 and V2 products (cf lanes 5, 7 and 8). Calcium ions (substituted for quinolone and Mg2+) also induced plasmid cleavage by topo IV but with differences in the spectrum of sites compared with that seen with gemifloxacin (Figure 5, cf lanes 5 and 9). E- and V-sites were both cleaved in the presence of Ca2+ and the same mutations at −2 and −4 positions that blocked drug-dependent DNA breakage (lanes 5–8) also inhibited Ca2+-promoted DNA scission (lanes 9–12). Blockade was not reversed by using supercoiled pEA1 as substrate (results not shown). Similar to quinolone cleavage, Ca2+ cleavage reactions were reversed by EDTA, salt and heat (results not shown). Overall, the results indicate that key determinants of quinolone and Ca2+ cleavage uncovered at the E-site also apply to the V-site validating the functional rules (Figure 3).Figure 6.


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

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

Gate mutations at E- and V-sites inhibit topo IV cleavage. ScaI-linearized plasmid pEA1 bearing wt or mutant E- and V-sites was incubated with topo IV alone (lanes 1–4) or in the presence of 2.5 µM gemifloxacin (lanes 5–8) or 32 mM Ca2+ (lanes 9–12). DNA cleavage was induced as described in previous figure legends and DNA products were examined by electrophoresis in a 1% agarose gel. Cleavage fragments V1 and V2, and E1 and E2, arising from breakage at the V- and E-sites are indicated by arrows. The presence of wt or mutant sites is indicated above each lane: −4C and −2T denote the presence of pairwise −4C/+8G or −2T/+6T mutations, respectively. M, DNA size markers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt696-F6: Gate mutations at E- and V-sites inhibit topo IV cleavage. ScaI-linearized plasmid pEA1 bearing wt or mutant E- and V-sites was incubated with topo IV alone (lanes 1–4) or in the presence of 2.5 µM gemifloxacin (lanes 5–8) or 32 mM Ca2+ (lanes 9–12). DNA cleavage was induced as described in previous figure legends and DNA products were examined by electrophoresis in a 1% agarose gel. Cleavage fragments V1 and V2, and E1 and E2, arising from breakage at the V- and E-sites are indicated by arrows. The presence of wt or mutant sites is indicated above each lane: −4C and −2T denote the presence of pairwise −4C/+8G or −2T/+6T mutations, respectively. M, DNA size markers.
Mentions: To compare topo IV action at E- and V-sites, we used linear pEA1 plasmid substrates bearing mutations at one or both sites in the presence of either 6 mM MgCl2 and gemifloxacin, or 32 mM Ca2+ ions (Figure 6). In the absence of drug or Ca2+, there was no topo IV-induced plasmid breakage (lanes 1–4). Inclusion of gemifloxacin promoted cleavage at a spectrum of sites, and as expected substitution of guanines at −4 with disfavoured cytosines abrogated gemifloxacin-stimulated cleavage at the E-site eliminating the E1 and E2 products (Figure 6, cf lanes 5 and 6). Introduction of −4C mutations or −2T changes on both strands of the V-site blocked cleavage at the site indicated by the absence of V1 and V2 products (cf lanes 5, 7 and 8). Calcium ions (substituted for quinolone and Mg2+) also induced plasmid cleavage by topo IV but with differences in the spectrum of sites compared with that seen with gemifloxacin (Figure 5, cf lanes 5 and 9). E- and V-sites were both cleaved in the presence of Ca2+ and the same mutations at −2 and −4 positions that blocked drug-dependent DNA breakage (lanes 5–8) also inhibited Ca2+-promoted DNA scission (lanes 9–12). Blockade was not reversed by using supercoiled pEA1 as substrate (results not shown). Similar to quinolone cleavage, Ca2+ cleavage reactions were reversed by EDTA, salt and heat (results not shown). Overall, the results indicate that key determinants of quinolone and Ca2+ cleavage uncovered at the E-site also apply to the V-site validating the functional rules (Figure 3).Figure 6.

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