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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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Chemical structure of CL.
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Figure 1: Chemical structure of CL.

Mentions: Clerocidin (CL) (Figure 1) is a cytotoxic diterpenoid natural product (1) that inhibits bacterial DNA gyrase and mammalian DNA topoisomerase II (2,3). Unlike other topoisomerase poisons, CL induces an irreversible DNA break when guanine (G) is present at the −1 position from the cleavage site (3–5). In the absence of topoisomerases, CL itself exhibits an intrinsic DNA alkylating activity against guanines that are exposed in single-stranded DNA regions, promoting spontaneous depurination and subsequent DNA scission at the abasic site (6). These observations and other data suggest that mammalian type II topoisomerases potentiate CL attack at guanines that have in some way been ‘processed’ in the enzyme-DNA complex (5,6). Thus, although toxicity has precluded clinical use, CL is of interest as a probe of the normally transient cleavage complex formed in the reaction cycle of type II topoisomerases.Figure 1.


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)

Chemical structure of CL.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Chemical structure of CL.
Mentions: Clerocidin (CL) (Figure 1) is a cytotoxic diterpenoid natural product (1) that inhibits bacterial DNA gyrase and mammalian DNA topoisomerase II (2,3). Unlike other topoisomerase poisons, CL induces an irreversible DNA break when guanine (G) is present at the −1 position from the cleavage site (3–5). In the absence of topoisomerases, CL itself exhibits an intrinsic DNA alkylating activity against guanines that are exposed in single-stranded DNA regions, promoting spontaneous depurination and subsequent DNA scission at the abasic site (6). These observations and other data suggest that mammalian type II topoisomerases potentiate CL attack at guanines that have in some way been ‘processed’ in the enzyme-DNA complex (5,6). Thus, although toxicity has precluded clinical use, CL is of interest as a probe of the normally transient cleavage complex formed in the reaction cycle of type II topoisomerases.Figure 1.

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