Molecular characterization of recombinant Pneumocystis carinii topoisomerase I: differential interactions with human topoisomerase I poisons and pentamidine.
The Pneumocystis carinii topoisomerase I-encoding gene has been cloned and sequenced, and the expressed enzyme interactions with several classes of topoisomerase I poisons have been characterized. The P. carinii topoisomerase I protein contains 763 amino acids and has a molecular mass of ca. 90 kDa. The expressed enzyme relaxes supercoiled DNA to completion and has no Mg2+ requirement. Cleavage assays reveal that both the human and P. carinii enzymes form covalent complexes in the presence of camptothecin, Hoechst 33342, and the terbenzimidazole QS-II-48. As with the human enzyme, no cleavage is stimulated in the presence of 4',6'-diamidino-2-phenylindole (DAPI) or berenil. A yeast cytotoxicity assay shows that P. carinii topoisomerase I is also a cytotoxic target for the mixed intercalative plus minor-groove binding drug nogalamycin. In contrast to the human enzyme, P. carinii topoisomerase I is resistant to both nitidine and potent protoberberine human topoisomerase I poisons. The differences in the sensitivities of P. carinii and human topoisomerase I to various topoisomerase I poisons support the use of the fungal enzyme as a molecular target for drug development. Additionally, we have characterized the interaction of pentamidine with P. carinii topoisomerase I. We show, by catalytic inhibition, cleavage, and yeast cytotoxicity assays, that pentamidine does not target topoisomerase I.
van Dross
Rukiyah T
RT
Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
Sanders
Marilyn M
MM
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D008969:Molecular Sequence Data; D010419:Pentamidine / Q000494:pharmacology*; D011010:Pneumocystis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D059004:Topoisomerase I Inhibitors
D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; D010419:Pentamidine; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin