Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.

摘要: Camptothecin (CPT) binds reversibly to, and thereby stabilizes, the cleavable complex formed between DNA topoisomerase I. The nature of interaction CPT with DNA-topoisomerase I binary was studied by use two affinity labeling reagents structurally related to camptothecin: 10-bromoacetamidomethylcamptothecin (BrCPT) 7-methyl-10-bromoacetamidomethylcamptothecin (BrCPTMe). These compounds have been shown trap irreversibly. Although cleavage plasmid mediated camptothecin reduced significantly treatment high salt or excess competitor DNA, enzyme-mediated stabilized BrCTPMe persisted for at least 4 h after similar treatment. production irreversible I-DNA time-dependent, suggesting that BrCPTMe first bound noncovalently enzyme-DNA and, in a second slower step, alkylated enzyme manner prevented ligation. formation covalent linkage supported experiments employed [3H]BrCPT, which label within complex. [3H]BrCPT enhanced greatly presence DNA; very little isolated occurred. Even inhibited camptothecin, both BrCPT same site on studies provide further evidence binding is created as suggest A-ring proximate an residue.