title: Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage.
abstract: Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the -1 position, i.e. immediately 5' of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.
authors.affiliation: Department of Pharmaceutical Sciences, University of Padova, 35131 Padova, Italy.
authors.forename: Sara N
authors.initials: SN
authors.lastname: Richter
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Leo
authors.forename: Giulia
authors.initials: G
authors.lastname: Giaretta
authors.forename: Barbara
authors.initials: B
authors.lastname: Gatto
authors.forename: L Mark
authors.initials: LM
authors.lastname: Fisher
authors.forename: Manlio
authors.initials: M
authors.lastname: Palumbo
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000633:toxicity*; D004249:DNA Damage; D027101:DNA Topoisomerase IV / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000633:toxicity; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000633:toxicity; D006147:Guanine / Q000032:analysis; D015363:Quinolones / Q000633:toxicity; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004224:Diterpenes; D004791:Enzyme Inhibitors; D015363:Quinolones; D006147:Guanine; C039027:clerocidin; D027101:DNA Topoisomerase IV