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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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Mutations at G79 and S81 in GyrA can discriminate gyrase poisoning by CL from that of quinolones. (A) Recombinant GyrA A79 and GyrA D79 proteins reconstitute a DNA supercoiling activity comparable to that of wild-type gyrase (wt). Relaxed pBR322 DNA was incubated with increasing amounts of wild-type or mutant S. pneumoniae GyrA subunits in the presence of 1 mM ATP and a fixed amount of wild type GyrB. DNA products were separated by electrophoresis in 1% agarose. (B, C and D) Differential poisoning of mutant gyrases by CL and CIP. Supercoiled pBR322 DNA was incubated with mutant gyrases (GyrA G79A, S81F or A79D) in the absence of ATP in a DNA cleavage assay containing CL or ciprofloxacin (CIP) at the concentrations indicated on the figure. In the four panels, N, R, L and SC denote nicked, relaxed, linear (EcoRI-cut) and supercoiled pBR322 DNA, respectively.
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Figure 7: Mutations at G79 and S81 in GyrA can discriminate gyrase poisoning by CL from that of quinolones. (A) Recombinant GyrA A79 and GyrA D79 proteins reconstitute a DNA supercoiling activity comparable to that of wild-type gyrase (wt). Relaxed pBR322 DNA was incubated with increasing amounts of wild-type or mutant S. pneumoniae GyrA subunits in the presence of 1 mM ATP and a fixed amount of wild type GyrB. DNA products were separated by electrophoresis in 1% agarose. (B, C and D) Differential poisoning of mutant gyrases by CL and CIP. Supercoiled pBR322 DNA was incubated with mutant gyrases (GyrA G79A, S81F or A79D) in the absence of ATP in a DNA cleavage assay containing CL or ciprofloxacin (CIP) at the concentrations indicated on the figure. In the four panels, N, R, L and SC denote nicked, relaxed, linear (EcoRI-cut) and supercoiled pBR322 DNA, respectively.

Mentions: In contrast to the DNA substrate, very little is known about the enzyme interactions that govern CL activity. The identification of a novel gyrA mutation (G79A) conferring CL-resistance in S. pneumoniae is therefore of considerable interest. The mutation affects a conserved glycine residue that lies in the CAP-like helix–turn–helix motif of GyrA thought to bind DNA (33,34). A different mutation of the equivalent glycine residue (to aspartate or cysteine) has been associated (albeit rarely) with clinical resistance to quinolones in E. coli (35,36). Moreover, in S. pneumoniae, adjacent mutations in GyrA of S81 to F or Y commonly cause resistance to quinolones (25,29,37) implying that a localized region of GyrA is implicated in binding different gyrase poisons. To understand better how CL interacts to poison gyrase, we expressed the S. pneumoniae G79A, S81F and G79D mutant GyrA subunits in E. coli and purified the proteins to homogeneity. In a supercoiling assay, the three mutant proteins reconstituted a gyrase activity comparable to that of the wild-type enzyme (Figure 7A, see ref. 29 for GyrA S81F data).Figure 7.


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)

Mutations at G79 and S81 in GyrA can discriminate gyrase poisoning by CL from that of quinolones. (A) Recombinant GyrA A79 and GyrA D79 proteins reconstitute a DNA supercoiling activity comparable to that of wild-type gyrase (wt). Relaxed pBR322 DNA was incubated with increasing amounts of wild-type or mutant S. pneumoniae GyrA subunits in the presence of 1 mM ATP and a fixed amount of wild type GyrB. DNA products were separated by electrophoresis in 1% agarose. (B, C and D) Differential poisoning of mutant gyrases by CL and CIP. Supercoiled pBR322 DNA was incubated with mutant gyrases (GyrA G79A, S81F or A79D) in the absence of ATP in a DNA cleavage assay containing CL or ciprofloxacin (CIP) at the concentrations indicated on the figure. In the four panels, N, R, L and SC denote nicked, relaxed, linear (EcoRI-cut) and supercoiled pBR322 DNA, respectively.
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Related In: Results  -  Collection

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Figure 7: Mutations at G79 and S81 in GyrA can discriminate gyrase poisoning by CL from that of quinolones. (A) Recombinant GyrA A79 and GyrA D79 proteins reconstitute a DNA supercoiling activity comparable to that of wild-type gyrase (wt). Relaxed pBR322 DNA was incubated with increasing amounts of wild-type or mutant S. pneumoniae GyrA subunits in the presence of 1 mM ATP and a fixed amount of wild type GyrB. DNA products were separated by electrophoresis in 1% agarose. (B, C and D) Differential poisoning of mutant gyrases by CL and CIP. Supercoiled pBR322 DNA was incubated with mutant gyrases (GyrA G79A, S81F or A79D) in the absence of ATP in a DNA cleavage assay containing CL or ciprofloxacin (CIP) at the concentrations indicated on the figure. In the four panels, N, R, L and SC denote nicked, relaxed, linear (EcoRI-cut) and supercoiled pBR322 DNA, respectively.
Mentions: In contrast to the DNA substrate, very little is known about the enzyme interactions that govern CL activity. The identification of a novel gyrA mutation (G79A) conferring CL-resistance in S. pneumoniae is therefore of considerable interest. The mutation affects a conserved glycine residue that lies in the CAP-like helix–turn–helix motif of GyrA thought to bind DNA (33,34). A different mutation of the equivalent glycine residue (to aspartate or cysteine) has been associated (albeit rarely) with clinical resistance to quinolones in E. coli (35,36). Moreover, in S. pneumoniae, adjacent mutations in GyrA of S81 to F or Y commonly cause resistance to quinolones (25,29,37) implying that a localized region of GyrA is implicated in binding different gyrase poisons. To understand better how CL interacts to poison gyrase, we expressed the S. pneumoniae G79A, S81F and G79D mutant GyrA subunits in E. coli and purified the proteins to homogeneity. In a supercoiling assay, the three mutant proteins reconstituted a gyrase activity comparable to that of the wild-type enzyme (Figure 7A, see ref. 29 for GyrA S81F data).Figure 7.

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