Previous studies have shown that formation of intermolecular DNA triplexes at sequences that overlap protein binding sites inhibits DNA binding by these proteins. We show that DNA cleavage by eukaryotic topoisomerase II is blocked by triplex formation at sites overlapping and adjacent to the triple binding site. To map precisely the boundaries of triplex interference, we constructed a vector containing enzyme binding sites of different lengths and flanked both 5` and 3` by DNA triplexes. We call this method Triplex Interference Mapping by Binding Element Replacement (TIMBER). Triplex regions within 3 bases 5` or 7 bases 3` of cleavage sites blocked DNA cleavage; triplex formation outside of this region had no effect upon cleavage activity. We conclude that topoisomerase II binding requires unhindered access to the major groove of a duplex DNA binding site in this 10-base region. In addition, the inclusion of topoisomerase II inhibitors yielded the same results for the triplex interference assays despite alterations in DNA cleavage site selection. The statistical analyses of over 500 topoisomerase II cleavage sites (in the presence or absence of inhibitors) suggest a model consistent with the region spanning -3 to +7 (relative to the cleavage site) containing most of the base-specific contacts for topoisomerase II. This triplex interference assay may prove valuable in the characterization of DNA binding sites for other proteins as well, particularly in conjunction with deletion analysis.

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