Influence of GC and AT specific DNA minor groove binding drugs on intermolecular triplex formation in the human c-Ki-ras promoter.

We have used DNase I footprinting and gel shift assays to characterize the interaction of DNA binding drugs mithramycin, distamycin, and berenil with an intermolecular triplex formed by the human c-Ki-ras promoter. A purine-rich triplex-forming oligonucleotide (ODN) forms a stable intermolecular triple helix (triplex) with a homopurine (PR):homopyrimidine (PY) motif in the human c-Ki-ras promoter which contains a 22bp PR:PY region (-328 to -307). This triplex structure is comprised of 15 G.G:C triplets interspersed with 7 T.A:T triplets. Mithramycin binding sites in the human c-Ki-ras promoter encompass most of the triplex target site and three G-C-rich sequences downstream of this triplex-forming region. Mithramycin binding within the c-Ki-ras promoter completely abrogates triplex formation. Furthermore, the addition of mithramycin to pre-formed triplex by c-Ki-ras promoter displaces the major groove bound ODN. Five prominent distamycin binding sites are noted within the c-Ki-ras promoter including the triplex-forming site as well as A-T-rich regions upstream and downstream of the triplex site. Berenil does not bind within the triplex target sequence, and only one berenil binding sequence downstream of the triplex motif was present within the c-Ki-ras promoter fragment. Neither distamycin nor berenil prevents triplex formation, and, furthermore, the addition of either distamycin or berenil to the pre-formed triplex structure did not displace the major-groove-bound third strand. This study demonstrates that GC-specific and AT-specific minor groove ligands differentially affect the intermolecular pur.pur:pyr triplex. A possible biological significance of mithramycin interaction with intramolecular triplex is discussed.