| Abstrakt: |
The cytotoxic alkaloid, camptothecin, does not inhibit the nicking-closing activity of the wheat germ type I topoisomerase (topo I). However, consistent with a previous report on the Hela cell topo I (Hsiang, Y.-H., Hertzberg, R., Hecht, S., and Liu, L. F. (1985) J. Biol. Chem. 260, 14873–14878), the drug does enhance DNA breakage when enzyme reactions are terminated with SDS. Drug-enhanced breakage was observed over the range of salt concentrations where the enzyme is most active (25–200 mMmonovalent cation). The presence of the drug did not appear to make the enzyme more processive in the range of salt concentrations from 100 to 170 mM, indicating that it probably does not affect the binding of the enzyme to DNA. Addition of high salt (0.5 M) to enzyme reactions containing camptothecin, prior to the addition of the detergent, prevented some, but not all of the drug-enhanced breakage. This result indicates that the drug causes some permanent, salt-stable nicking of the DNA, an observation that may explain its cytotoxic effects. A comparison of the breakage specificity in the presence of the drug with the consensus sequence for breakage determined previously (Been, M. D., Burgess, R. R., and Champoux, J. J. (1984) Nucleic Acids Res.12, 3097–3114) indicated that the drug has a minimal effect on the sequence specificity of the enzyme. However, the drug enhanced breakage at different sites to quite different extents. Therefore, camptothecin should be useful for localizing topo I break sites in vivo, but quantitative comparisons on the relative frequencies of breakage at different locations should be avoided. |
