Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage.
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.
Pan
Xiao Su
XS
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.
Dias
Miriam
M
Palumbo
Manlio
M
Fisher
L Mark
LM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000633:toxicity*; D001483:Base Sequence; D004249:DNA Damage; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000633:toxicity; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000633:toxicity*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017422:Sequence Analysis, DNA; D013296:Streptococcus pneumoniae / Q000201:enzymology; D059005:Topoisomerase II Inhibitors
D000900:Anti-Bacterial Agents; D004278:DNA, Superhelical; D004224:Diterpenes; D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; C039027:clerocidin; D000255:Adenosine Triphosphate; D027081:DNA Gyrase