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 whi ch DNA strands are cleaved and covalently linked to the enzyme. In thi s work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated v ariants were used to determine the effects of base mutations on DNA cl eavage levels produced by murine topoisomerase II with and without ida rubicin. Although site competition could affect cleavage levels, mutat ion effects were rather similar among several cleavage sites. The majo r 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 f or DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzym e binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results in dicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cle aved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracy clines. An important finding was that mutations that improve the fit t o the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively inter act with one enzyme subunit only and trap the whole dimeric enzyme. Th ese findings documented that DNA recognition by topoisomerase II may o ccur 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 hel ix.