Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage.
Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.
Bigioni
M
M
Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Zunino
F
F
Capranico
G
G
D000225:Adenine / Q000378:metabolism; D001482:Base Composition; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D015255:Idarubicin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation
D004247:DNA; D004250:DNA Topoisomerases, Type II; D000225:Adenine; D015255:Idarubicin