Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms α and β

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for a. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase I1β (overexpressed and purified from yeast) and a direct comparison with the recombinant α isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by α or β in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human α promoted DNA breakage at sites akin to those of β, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for β and 70 sites for α revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant α isoform is in agreement with previous studies using α purified from human cell lines. Thus, α and β proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II α; the results presented here demonstrate that β is an in vitro target with similar site determinants, strongly suggesting that β should also be considered a target of mAMSA in vivo.