Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site.
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.
Hertzberg
R P
RP
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Busby
R W
RW
Caranfa
M J
MJ
Holden
K G
KG
Johnson
R K
RK
Hecht
S M
SM
Kingsbury
W D
WD
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
D000345:Affinity Labels; C066310:10-bromoacetamidomethylcamptothecin; C066311:7-methyl-10-bromoacetamidomethylcamptothecin; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin