Gee J E, Blume S, Snyder R C, Ray R, Miller D M
Department of Biochemistry, University of Alabama, Birmingham 35294.
J Biol Chem. 1992 Jun 5;267(16):11163-7.
The human dihydrofolate reductase (DHFR) promoter sequence contains two consensus binding sites for the Sp1 regulatory protein. We have determined the effect of intermolecular triplex DNA formation on Sp1 binding to the DHFR promoter. The DHFR Sp1 binding site I (-39 to -48 relative to the DHFR transcription start site) demonstrates concentration-dependent triplex formation with a 19-base pair G-rich oligonucleotide (GR19) which is complementary to the polypyrimidine strand. DNase I footprint analysis demonstrates that GR19 forms a DNA triplex structure with the DHFR promoter fragment in a sequence-specific manner. DNase I footprinting analysis also indicates that the orientation of binding of these G-rich oligonucleotides is antiparallel. CR19, a C-rich complementary oligonucleotide, on the other hand, does not form triplex. The DNase I protection pattern of DHFR promoter fragment incubated with both recombinant Sp1 and triplex-forming oligonucleotide suggests that triplex formation prevents Sp1 binding. This is confirmed by gel shift analysis which demonstrates that triplex formation by the Sp1 binding sequences of the DHFR promoter prevents recombinant Sp1 binding in a concentration-dependent manner. These results demonstrate that intermolecular triplex formation prevents regulatory protein binding in a sequence-specific manner.
人类二氢叶酸还原酶（DHFR）启动子序列包含Sp1调节蛋白的两个共有结合位点。我们已经确定了分子间三链体DNA形成对Sp1与DHFR启动子结合的影响。DHFR Sp1结合位点I（相对于DHFR转录起始位点为-39至-48）与富含鸟嘌呤的19个碱基对寡核苷酸（GR19）呈现浓度依赖性三链体形成，GR19与多嘧啶链互补。DNase I足迹分析表明，GR19以序列特异性方式与DHFR启动子片段形成DNA三链体结构。DNase I足迹分析还表明，这些富含鸟嘌呤的寡核苷酸的结合方向是反平行的。另一方面，富含胞嘧啶的互补寡核苷酸CR19不形成三链体。与重组Sp1和三链体形成寡核苷酸一起孵育的DHFR启动子片段的DNase I保护模式表明，三链体形成会阻止Sp1结合。凝胶迁移分析证实了这一点，该分析表明，DHFR启动子的Sp1结合序列形成的三链体以浓度依赖性方式阻止重组Sp1结合。这些结果表明，分子间三链体形成以序列特异性方式阻止调节蛋白结合。
