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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-stimulated gyrase cleavage at the pBR322 ‘1073’ site. (A) DNA modification and EDTA reversal. A 234-bp pBR322 fragment (nucleotides 966–1200), labelled with 33P at the 5′ end of the TOP or BOTTOM strand, was incubated with gyrase and 1 mM ATP in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (Gm) (lanes 8 and 9). Samples in lanes 5–7 were further incubated with NaCl (S), EDTA (E) or at 65°C (T) as described in the Figure 4 legend. After treatment with SDS and proteinase K, reaction products were precipitated with ethanol. Samples in lanes 4–7 and 9 were incubated with hot piperidine. Samples were lyophilized and run on 6% denaturing polyacrylamide gels alongside G+A chemical sequencing and ACGT chain termination sequencing products. Lanes 1 and 2 are controls for untreated DNA and gyrase processed DNA, respectively. Lanes 3 and 8 are non-piperidine treated cleavage products. The main cleavage site at 1073 produced CL-stabilized fragments indicated by a filled circle (TOP strand, piperidine-shifted and irreversible) and by an unfilled circle (BOTTOM strand, largely unshifted by piperidine and EDTA reversible). (B) Location of DNA gyrase cleavage sites in the pBR322 fragment. Symbols are used as described in the Figure 4B legend. Large arrowheads denote the ‘1073’ site; smaller arrowheads indicate weaker sites. Nucleotide sequence from ref 30.
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Figure 5: CL-stimulated gyrase cleavage at the pBR322 ‘1073’ site. (A) DNA modification and EDTA reversal. A 234-bp pBR322 fragment (nucleotides 966–1200), labelled with 33P at the 5′ end of the TOP or BOTTOM strand, was incubated with gyrase and 1 mM ATP in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (Gm) (lanes 8 and 9). Samples in lanes 5–7 were further incubated with NaCl (S), EDTA (E) or at 65°C (T) as described in the Figure 4 legend. After treatment with SDS and proteinase K, reaction products were precipitated with ethanol. Samples in lanes 4–7 and 9 were incubated with hot piperidine. Samples were lyophilized and run on 6% denaturing polyacrylamide gels alongside G+A chemical sequencing and ACGT chain termination sequencing products. Lanes 1 and 2 are controls for untreated DNA and gyrase processed DNA, respectively. Lanes 3 and 8 are non-piperidine treated cleavage products. The main cleavage site at 1073 produced CL-stabilized fragments indicated by a filled circle (TOP strand, piperidine-shifted and irreversible) and by an unfilled circle (BOTTOM strand, largely unshifted by piperidine and EDTA reversible). (B) Location of DNA gyrase cleavage sites in the pBR322 fragment. Symbols are used as described in the Figure 4B legend. Large arrowheads denote the ‘1073’ site; smaller arrowheads indicate weaker sites. Nucleotide sequence from ref 30.

Mentions: Reversible and irreversible DNA scission by CL with covalent modification at some sites was also seen using a different DNA substrate, namely a PCR product amplified from plasmid pBR322 (nucleotides 966–1200) and 5′-33P end-labelled in the TOP or BOTTOM strands (Figure 5A).Figure 5.


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-stimulated gyrase cleavage at the pBR322 ‘1073’ site. (A) DNA modification and EDTA reversal. A 234-bp pBR322 fragment (nucleotides 966–1200), labelled with 33P at the 5′ end of the TOP or BOTTOM strand, was incubated with gyrase and 1 mM ATP in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (Gm) (lanes 8 and 9). Samples in lanes 5–7 were further incubated with NaCl (S), EDTA (E) or at 65°C (T) as described in the Figure 4 legend. After treatment with SDS and proteinase K, reaction products were precipitated with ethanol. Samples in lanes 4–7 and 9 were incubated with hot piperidine. Samples were lyophilized and run on 6% denaturing polyacrylamide gels alongside G+A chemical sequencing and ACGT chain termination sequencing products. Lanes 1 and 2 are controls for untreated DNA and gyrase processed DNA, respectively. Lanes 3 and 8 are non-piperidine treated cleavage products. The main cleavage site at 1073 produced CL-stabilized fragments indicated by a filled circle (TOP strand, piperidine-shifted and irreversible) and by an unfilled circle (BOTTOM strand, largely unshifted by piperidine and EDTA reversible). (B) Location of DNA gyrase cleavage sites in the pBR322 fragment. Symbols are used as described in the Figure 4B legend. Large arrowheads denote the ‘1073’ site; smaller arrowheads indicate weaker sites. Nucleotide sequence from ref 30.
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Related In: Results  -  Collection

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Figure 5: CL-stimulated gyrase cleavage at the pBR322 ‘1073’ site. (A) DNA modification and EDTA reversal. A 234-bp pBR322 fragment (nucleotides 966–1200), labelled with 33P at the 5′ end of the TOP or BOTTOM strand, was incubated with gyrase and 1 mM ATP in the absence (lane 2) or presence of 200 μM CL (lanes 3–7) or 100 μM gemifloxacin (Gm) (lanes 8 and 9). Samples in lanes 5–7 were further incubated with NaCl (S), EDTA (E) or at 65°C (T) as described in the Figure 4 legend. After treatment with SDS and proteinase K, reaction products were precipitated with ethanol. Samples in lanes 4–7 and 9 were incubated with hot piperidine. Samples were lyophilized and run on 6% denaturing polyacrylamide gels alongside G+A chemical sequencing and ACGT chain termination sequencing products. Lanes 1 and 2 are controls for untreated DNA and gyrase processed DNA, respectively. Lanes 3 and 8 are non-piperidine treated cleavage products. The main cleavage site at 1073 produced CL-stabilized fragments indicated by a filled circle (TOP strand, piperidine-shifted and irreversible) and by an unfilled circle (BOTTOM strand, largely unshifted by piperidine and EDTA reversible). (B) Location of DNA gyrase cleavage sites in the pBR322 fragment. Symbols are used as described in the Figure 4B legend. Large arrowheads denote the ‘1073’ site; smaller arrowheads indicate weaker sites. Nucleotide sequence from ref 30.
Mentions: Reversible and irreversible DNA scission by CL with covalent modification at some sites was also seen using a different DNA substrate, namely a PCR product amplified from plasmid pBR322 (nucleotides 966–1200) and 5′-33P end-labelled in the TOP or BOTTOM strands (Figure 5A).Figure 5.

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