In vitro evolution of preferred topoisomerase II DNA cleavage sites

Topoisomerase II is an essential enzyme that is the target for several clin ically important anticancer drugs. Although this enzyme must create transie nt double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its n ucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme specificity, a modified systematic evolution of li gands by exponential enrichment (SELEX) protocol was employed to select/evo lve DNA sequences that were preferentially cleaved by Drosophila melanogast er topoisomerase II. Levels of DNA scission rose substantially (from 3 to 2 0%) over 20 rounds of SELEX, In vitro selection/evolution converged on an a lternating purine/pyrmidine sequence that was highly AT-rich (TATATATACATAT ATATA). The preference for this sequence was more pronounced for Drosophila topoisomerase II over other species and was increased in the presence of D NA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be ba sed on higher rates of DNA scission rather than increased binding affinity or decreased religation rates. The preferred sequence for topoisomerase II- mediated DNA cleavage is dramatically overrepresented (similar to 10,000-fo ld) in the euchromatic genome of D. melanogaster, implying that it may be a site for the physiological action of this enzyme.