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Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.

Pan XS, Dias M, Palumbo M, Fisher LM - Nucleic Acids Res. (2008)

Bottom Line: CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci.Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones.The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

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CL inhibits DNA gyrase and stimulates enzyme-mediated DNA cleavage. (A) Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 DNA was incubated with S. pneumoniae gyrase (1U) and 1.4 mM ATP in the absence or presence of CL or the quinolone ciprofloxacin (CIP) at the concentrations indicated on the figure. Reaction mixtures were loaded on a 1% agarose gel and DNA bands were visualized by staining with ethidium bromide and uv light. Lane a, supercoiled pBR322 (SC); lane b, relaxed pBR322 (R). (B) Stimulation of gyrase-mediated DNA breakage. Supercoiled pBR322 was incubated at 37°C for 1 h with gyrase (in the absence of ATP) with CL or CIP at the concentrations indicated on the figure. After addition of SDS and incubation with proteinase K, DNA products were separated and displayed by electrophoresis in 1% agarose. (Lane a) is the input supercoiled pBR322, (lane b) is pBR322 linearized with EcoRI. N, L and SC denote nicked, linear and supercoiled plasmid DNA, respectively. In (A) and (B), gel images were captured using an Alpha Innotech digital camera and DNA bands were quantitated and analysed using associated software.
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Figure 2: CL inhibits DNA gyrase and stimulates enzyme-mediated DNA cleavage. (A) Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 DNA was incubated with S. pneumoniae gyrase (1U) and 1.4 mM ATP in the absence or presence of CL or the quinolone ciprofloxacin (CIP) at the concentrations indicated on the figure. Reaction mixtures were loaded on a 1% agarose gel and DNA bands were visualized by staining with ethidium bromide and uv light. Lane a, supercoiled pBR322 (SC); lane b, relaxed pBR322 (R). (B) Stimulation of gyrase-mediated DNA breakage. Supercoiled pBR322 was incubated at 37°C for 1 h with gyrase (in the absence of ATP) with CL or CIP at the concentrations indicated on the figure. After addition of SDS and incubation with proteinase K, DNA products were separated and displayed by electrophoresis in 1% agarose. (Lane a) is the input supercoiled pBR322, (lane b) is pBR322 linearized with EcoRI. N, L and SC denote nicked, linear and supercoiled plasmid DNA, respectively. In (A) and (B), gel images were captured using an Alpha Innotech digital camera and DNA bands were quantitated and analysed using associated software.

Mentions: Given the outcome of the genetic experiments, we tested the effects of CL on S. pneumoniae gyrase in catalytic and DNA cleavage assays (Figure 2). As a control, we employed ciprofloxacin, a fluoroquinolone often used as a comparator for inhibition/cleavage studies. In the absence of drug (Figure 2A, lanes 0), gyrase converted relaxed plasmid DNA (lane b) into the supercoiled form in an ATP-dependent reaction. CL inhibited this activity in a dose-dependent fashion with 50% inhibition (IC50) at 40 μM, i.e. comparable to the ciprofloxacin IC50 of ∼80 μM (Figure 2A, compare left and right lanes).Figure 2.


Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.

Pan XS, Dias M, Palumbo M, Fisher LM - Nucleic Acids Res. (2008)

CL inhibits DNA gyrase and stimulates enzyme-mediated DNA cleavage. (A) Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 DNA was incubated with S. pneumoniae gyrase (1U) and 1.4 mM ATP in the absence or presence of CL or the quinolone ciprofloxacin (CIP) at the concentrations indicated on the figure. Reaction mixtures were loaded on a 1% agarose gel and DNA bands were visualized by staining with ethidium bromide and uv light. Lane a, supercoiled pBR322 (SC); lane b, relaxed pBR322 (R). (B) Stimulation of gyrase-mediated DNA breakage. Supercoiled pBR322 was incubated at 37°C for 1 h with gyrase (in the absence of ATP) with CL or CIP at the concentrations indicated on the figure. After addition of SDS and incubation with proteinase K, DNA products were separated and displayed by electrophoresis in 1% agarose. (Lane a) is the input supercoiled pBR322, (lane b) is pBR322 linearized with EcoRI. N, L and SC denote nicked, linear and supercoiled plasmid DNA, respectively. In (A) and (B), gel images were captured using an Alpha Innotech digital camera and DNA bands were quantitated and analysed using associated software.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: CL inhibits DNA gyrase and stimulates enzyme-mediated DNA cleavage. (A) Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 DNA was incubated with S. pneumoniae gyrase (1U) and 1.4 mM ATP in the absence or presence of CL or the quinolone ciprofloxacin (CIP) at the concentrations indicated on the figure. Reaction mixtures were loaded on a 1% agarose gel and DNA bands were visualized by staining with ethidium bromide and uv light. Lane a, supercoiled pBR322 (SC); lane b, relaxed pBR322 (R). (B) Stimulation of gyrase-mediated DNA breakage. Supercoiled pBR322 was incubated at 37°C for 1 h with gyrase (in the absence of ATP) with CL or CIP at the concentrations indicated on the figure. After addition of SDS and incubation with proteinase K, DNA products were separated and displayed by electrophoresis in 1% agarose. (Lane a) is the input supercoiled pBR322, (lane b) is pBR322 linearized with EcoRI. N, L and SC denote nicked, linear and supercoiled plasmid DNA, respectively. In (A) and (B), gel images were captured using an Alpha Innotech digital camera and DNA bands were quantitated and analysed using associated software.
Mentions: Given the outcome of the genetic experiments, we tested the effects of CL on S. pneumoniae gyrase in catalytic and DNA cleavage assays (Figure 2). As a control, we employed ciprofloxacin, a fluoroquinolone often used as a comparator for inhibition/cleavage studies. In the absence of drug (Figure 2A, lanes 0), gyrase converted relaxed plasmid DNA (lane b) into the supercoiled form in an ATP-dependent reaction. CL inhibited this activity in a dose-dependent fashion with 50% inhibition (IC50) at 40 μM, i.e. comparable to the ciprofloxacin IC50 of ∼80 μM (Figure 2A, compare left and right lanes).Figure 2.

Bottom Line: CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci.Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones.The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK.

ABSTRACT
Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.

Show MeSH
Related in: MedlinePlus