title: Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.
abstract: Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA and topoisomerase I. The nature of the interaction of CPT with the DNA-topoisomerase I binary complex was studied by the use of two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) and 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown to trap the DNA-topoisomerase I complex irreversibly. Although cleavage of DNA plasmid mediated by topoisomerase I and camptothecin was reduced significantly by treatment with high salt or excess competitor DNA, enzyme-mediated DNA cleavage stabilized by BrCTPMe persisted for at least 4 h after similar treatment. The production of irreversible topoisomerase I-DNA cleavage was time-dependent, suggesting that BrCPTMe first bound noncovalently to the enzyme-DNA complex and, in a second slower step, alkylated the enzyme or DNA in a manner that prevented DNA ligation. The formation of a covalent linkage was supported by experiments that employed [3H]BrCPT, which was shown to label topoisomerase I within the enzyme-DNA complex. [3H]BrCPT labeling of topoisomerase I was enhanced greatly by the presence of DNA; very little labeling of isolated topoisomerase I or isolated DNA occurred. Even in the presence of DNA, [3H]BrCPT labeling of topoisomerase I was inhibited by camptothecin, suggesting that both CPT and BrCPT bound to the same site on the DNA-topoisomerase I binary complex. These studies provide further evidence that a binding site for camptothecin is created as the DNA-topoisomerase I complex is formed and suggest that the A-ring of camptothecin is proximate to an enzyme residue.
authors.affiliation: Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: R P
authors.initials: RP
authors.lastname: Hertzberg
authors.forename: R W
authors.initials: RW
authors.lastname: Busby
authors.forename: M J
authors.initials: MJ
authors.lastname: Caranfa
authors.forename: K G
authors.initials: KG
authors.lastname: Holden
authors.forename: R K
authors.initials: RK
authors.lastname: Johnson
authors.forename: S M
authors.initials: SM
authors.lastname: Hecht
authors.forename: W D
authors.initials: WD
authors.lastname: Kingsbury
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
chemical_list: D000345:Affinity Labels; C066310:10-bromoacetamidomethylcamptothecin; C066311:7-methyl-10-bromoacetamidomethylcamptothecin; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin