Vigneswaran N, Mayfield C A, Rodu B, James R, Kim H G, Miller D M
Department of Oral Pathology, Bolden Laboratory, University of Alabama at Birmingham 35294-3300, USA.
Biochemistry. 1996 Jan 30;35(4):1106-14. doi: 10.1021/bi951562b.
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.
我们运用DNA酶I足迹法和凝胶迁移实验来表征DNA结合药物光神霉素、偏端霉素和贝尼尔与人c-Ki-ras启动子形成的分子间三链体的相互作用。富含嘌呤的三链体形成寡核苷酸（ODN）与人c-Ki-ras启动子中含22bp嘌呤（PR）：嘧啶（PY）区域（-328至-307）的同型嘌呤（PR）：同型嘧啶（PY）基序形成稳定的分子间三链螺旋（三链体）。该三链体结构由15个G.G:C三联体和7个T.A:T三联体相间组成。人c-Ki-ras启动子中的光神霉素结合位点涵盖了大部分三链体靶位点以及该三链体形成区域下游的三个富含G-C的序列。光神霉素在c-Ki-ras启动子内的结合完全消除了三链体的形成。此外，将光神霉素添加到由c-Ki-ras启动子预先形成的三链体中会取代与大沟结合的ODN。在c-Ki-ras启动子内发现了五个显著的偏端霉素结合位点，包括三链体形成位点以及三链体位点上游和下游的富含A-T的区域。贝尼尔不在三链体靶序列内结合，并且在c-Ki-ras启动子片段中仅存在三链体基序下游的一个贝尼尔结合序列。偏端霉素和贝尼尔都不会阻止三链体的形成，此外，将偏端霉素或贝尼尔添加到预先形成的三链体结构中不会取代与大沟结合的第三条链。本研究表明，GC特异性和AT特异性小沟配体对分子间嘌呤·嘌呤：嘧啶三链体有不同影响。讨论了光神霉素与分子内三链体相互作用的可能生物学意义。
