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J Biol Chem, Vol. 274, Issue 8, 5227-5235, February 19, 1999
From the Departments of Topoisomerase II is an essential enzyme that is
the target for several clinically important anticancer drugs. Although
this enzyme must create transient double-stranded breaks in the genetic material in order to carry out its indispensable DNA strand passage reaction, the factors that underlie its nucleotide cleavage specificity remain an enigma. Therefore, to address the critical issue of enzyme
specificity, a modified systematic evolution of ligands by exponential
enrichment (SELEX) protocol was employed to select/evolve DNA sequences
that were preferentially cleaved by Drosophila melanogaster topoisomerase II. Levels of DNA scission rose substantially (from 3 to
20%) over 20 rounds of SELEX. In vitro selection/evolution converged on an alternating purine/pyrmidine sequence that was highly
AT-rich (TATATATACATATATATA). The preference for this sequence was more
pronounced for Drosophila topoisomerase II over other species and was increased in the presence of DNA cleavage-enhancing anticancer drugs. Enhanced cleavage appeared to be based 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
(~10,000-fold) in the euchromatic genome of D. melanogaster, implying that it may be a site for the
physiological action of this enzyme.
In Vitro Evolution of Preferred Topoisomerase II
DNA Cleavage Sites
and
¶
Biochemistry and
¶ Medicine (Oncology), Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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