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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-promoted DNA cleavage mediated by gyrase occurs at specific sites and is stimulated by ATP. The 290-bp S fragment (from the S. pneumoniae parE gene) 33P-labelled at the 5′ end of the TOP or BOTTOM strand was cleaved by S. pneumoniae gyrase in the absence or presence of 200 μM CL or 100 μM gemifloxacin (Gemi) and 1 mM ATP. These drug concentrations were chosen so as to achieve comparable cleavage levels of the linear DNA substrate. DNA products were separated by electrophoresis in a denaturing 6% polyacrylamide gel alongside DNA sequencing products (ACGT) obtained by the chain termination method using the same 5′-labelled oligonucleotide primers employed in PCR.
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Figure 3: CL-promoted DNA cleavage mediated by gyrase occurs at specific sites and is stimulated by ATP. The 290-bp S fragment (from the S. pneumoniae parE gene) 33P-labelled at the 5′ end of the TOP or BOTTOM strand was cleaved by S. pneumoniae gyrase in the absence or presence of 200 μM CL or 100 μM gemifloxacin (Gemi) and 1 mM ATP. These drug concentrations were chosen so as to achieve comparable cleavage levels of the linear DNA substrate. DNA products were separated by electrophoresis in a denaturing 6% polyacrylamide gel alongside DNA sequencing products (ACGT) obtained by the chain termination method using the same 5′-labelled oligonucleotide primers employed in PCR.

Mentions: The specificity of gyrase-mediated DNA breakage promoted by CL was examined using the 5′-end 33P-labelled S fragment from the S. pneumoniae parE gene and compared to that of gemifloxacin, an anti-pneumococcal quinolone whose induction of sequence-specific cleavage has been studied in detail for pneumococcal gyrase and topo IV (31). Cleavage of TOP and BOTTOM DNA strands by both drugs was markedly enhanced by the inclusion of ATP and required the presence of gyrase: no breakage was seen with CL alone consistent with the known poor reactivity of CL toward duplex DNA (Figure 3, Introduction section). CL stimulated a unique repertoire of cuts, though some sites were also cleaved with gemifloxacin (Figure 3). Quinolones are known to stabilize DNA cleavage fragments that possess 3′ OH ends and hence co-migrate with chain termination DNA sequencing products (21,31). CL-generated fragments also co-migrated with dideoxy sequencing products (and some quinolone-stimulated fragments) (Figure 3 and later Figures 4–6) allowing their mapping at the sequence level. CL-induced gyrase scission occurred either at single sites or more frequently in pairs on complementary strands showing the characteristic 4-bp stagger expected of double-stranded DNA breakage by gyrase (31). A compilation of sites mapped in the pneumococcal S fragment or plasmid pBR322 (see below) showed a strong preference for guanine at the −1 position or the equivalent C at +5 (Table 1). Ten sites of double-stranded DNA cleavage had both −1G and +5C, i.e. guanine at both −1 positions. This is the first sequence analysis of CL-promoted cleavage by gyrase.Figure 3.


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-promoted DNA cleavage mediated by gyrase occurs at specific sites and is stimulated by ATP. The 290-bp S fragment (from the S. pneumoniae parE gene) 33P-labelled at the 5′ end of the TOP or BOTTOM strand was cleaved by S. pneumoniae gyrase in the absence or presence of 200 μM CL or 100 μM gemifloxacin (Gemi) and 1 mM ATP. These drug concentrations were chosen so as to achieve comparable cleavage levels of the linear DNA substrate. DNA products were separated by electrophoresis in a denaturing 6% polyacrylamide gel alongside DNA sequencing products (ACGT) obtained by the chain termination method using the same 5′-labelled oligonucleotide primers employed in PCR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553588&req=5

Figure 3: CL-promoted DNA cleavage mediated by gyrase occurs at specific sites and is stimulated by ATP. The 290-bp S fragment (from the S. pneumoniae parE gene) 33P-labelled at the 5′ end of the TOP or BOTTOM strand was cleaved by S. pneumoniae gyrase in the absence or presence of 200 μM CL or 100 μM gemifloxacin (Gemi) and 1 mM ATP. These drug concentrations were chosen so as to achieve comparable cleavage levels of the linear DNA substrate. DNA products were separated by electrophoresis in a denaturing 6% polyacrylamide gel alongside DNA sequencing products (ACGT) obtained by the chain termination method using the same 5′-labelled oligonucleotide primers employed in PCR.
Mentions: The specificity of gyrase-mediated DNA breakage promoted by CL was examined using the 5′-end 33P-labelled S fragment from the S. pneumoniae parE gene and compared to that of gemifloxacin, an anti-pneumococcal quinolone whose induction of sequence-specific cleavage has been studied in detail for pneumococcal gyrase and topo IV (31). Cleavage of TOP and BOTTOM DNA strands by both drugs was markedly enhanced by the inclusion of ATP and required the presence of gyrase: no breakage was seen with CL alone consistent with the known poor reactivity of CL toward duplex DNA (Figure 3, Introduction section). CL stimulated a unique repertoire of cuts, though some sites were also cleaved with gemifloxacin (Figure 3). Quinolones are known to stabilize DNA cleavage fragments that possess 3′ OH ends and hence co-migrate with chain termination DNA sequencing products (21,31). CL-generated fragments also co-migrated with dideoxy sequencing products (and some quinolone-stimulated fragments) (Figure 3 and later Figures 4–6) allowing their mapping at the sequence level. CL-induced gyrase scission occurred either at single sites or more frequently in pairs on complementary strands showing the characteristic 4-bp stagger expected of double-stranded DNA breakage by gyrase (31). A compilation of sites mapped in the pneumococcal S fragment or plasmid pBR322 (see below) showed a strong preference for guanine at the −1 position or the equivalent C at +5 (Table 1). Ten sites of double-stranded DNA cleavage had both −1G and +5C, i.e. guanine at both −1 positions. This is the first sequence analysis of CL-promoted cleavage by gyrase.Figure 3.

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