Effects of antineoplastic drugs on the post-strand-passage DNA cleavage/religation equilibrium of topoisomerase II.
The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.
Robinson
M J
MJ
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Osheroff
N
N
D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology*; D000677:Amsacrine / Q000494:pharmacology*; D000818:Animals; D004247:DNA / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D005047:Etoposide / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics
D000677:Amsacrine; D000266:Adenylyl Imidodiphosphate; D005047:Etoposide; D004247:DNA; D004250:DNA Topoisomerases, Type II