Drug-promoted cleavage of kinetoplast DNA minicircles. Evidence for type II topoisomerase activity in trypanosome mitochondria.
Minicircle DNA, the major component of the mitochondrial DNA of trypanosomes (kinetoplast DNA), is linearized when living Trypanosoma equiperdum cells are treated with inhibitors of mammalian type II topoisomerases and then lysed with sodium dodecyl sulfate. A variety of intercalating and nonintercalating compounds (the epipodophyllotoxins, 4'-(9-acridinylamino)-methanesulfon-m-anisidine, 2-methyl-9-hydroxyellipticine, and acriflavine) are active, but novobiocin and specific gyrase inhibitors (the quinolones) are not. The linearized minicircles are in a DNA-protein complex, as their electrophoretic mobility is increased by Proteinase K treatment. They are digested by exonuclease III but not by lambda exonuclease, indicating that the protein must be linked to both 5' ends. Drug-induced cleavage sites vary with different compounds and are found throughout the minicircle sequence. These results indicate that trypanosome mitochondria contain a type II topoisomerase with some properties similar to those of type II topoisomerases in the nucleus of higher eukaryotes. A maximum of 12% of all minicircles is cleaved in the presence of VP16-213, indicating there are at least 600 molecules of mitochondrial type II topoisomerase/cell or about one enzyme/8 kilobases of minicircle DNA.
Shapiro
T A
TA
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Klein
V A
VA
Englund
P T
PT
D000818:Animals; D002874:Chromosome Mapping; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004270:DNA, Circular / Q000187:drug effects* / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000187:drug effects*; D005047:Etoposide / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D059005:Topoisomerase II Inhibitors; D014345:Trypanosoma / Q000201:enzymology*
D004270:DNA, Circular; D004272:DNA, Mitochondrial; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D010447:Peptide Hydrolases; D004250:DNA Topoisomerases, Type II