Li Wei, Miyoshi Daisuke, Nakano Shu-ichi, Sugimoto Naoki
High Technology Research Center, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan.
Biochemistry. 2003 Oct 14;42(40):11736-44. doi: 10.1021/bi034168j.
Structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex has been implicated for repetitive DNA sequences, but the competitive mechanism of these multistranded structures still needs to be elucidated. We investigated the effects of sequence context, cation species, and pH on duplex formation by the G-quadruplex of dG(3)(T(2)AG(3))(3) and its complement the I-motif of d(C(3)TA(2))(3)C(3), using ITC, DSC, PAGE, CD, UV, and CD stopped-flow kinetic techniques. ITC and PAGE experiments confirmed Watson-Crick duplex formation by the complementary strands. The binding constant of the two DNA strands in the presence of 10 mM Mg(2+) at pH 7.0 was shown to be 5.28 x 10(7) M(-1) at 20 degrees C, about 400 times larger than that in the presence of 100 mM Na(+) at pH 5.5. The dynamic transition traces of the duplex formation from the equimolar mixture of G-/C-rich complementary sequences were obtained at both pH 7.0 and pH 5.5. Fitting to a single-exponential function gave an observed rate of 8.06 x 10(-3) s(-1) at 20 degrees C in 10 mM Mg(2+) buffer at pH 7.0, which was about 10 times the observed rate at pH 5.5 under the same conditions. Both of the observed rates increased as temperature rose, implying that the dissociation of the single-stranded structured DNAs is the rate-limiting step for the WC duplex formation. The difference between the apparent activation energy at pH 7.0 and that at pH 5.5 reflects the fact that pH significantly influences the structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex, which also implies a possible biological role for I-motifs in biological regulation.
G-四链体、I-基序和沃森-克里克双链体之间的结构竞争与重复DNA序列有关，但这些多链结构的竞争机制仍有待阐明。我们使用等温滴定量热法（ITC）、差示扫描量热法（DSC）、聚丙烯酰胺凝胶电泳（PAGE）、圆二色光谱（CD）、紫外光谱（UV）和CD停流动力学技术，研究了序列背景、阳离子种类和pH对dG(3)(T(2)AG(3))(3)的G-四链体及其互补链d(C(3)TA(2))(3)C(3)的I-基序形成双链体的影响。ITC和PAGE实验证实了互补链形成了沃森-克里克双链体。在20℃、pH 7.0条件下，10 mM Mg(2+)存在时两条DNA链的结合常数为5.28×10(7) M(-1)，约为pH 5.5、100 mM Na(+)存在时结合常数的400倍。在pH 7.0和pH 5.5条件下，均获得了富含G/C的互补序列等摩尔混合物形成双链体的动态转变轨迹。在20℃、pH 7.0的10 mM Mg(2+)缓冲液中，拟合为单指数函数得到的观测速率为8.06×10(-3) s(-1)，约为相同条件下pH 5.5时观测速率的10倍。随着温度升高，两个观测速率均增加，这意味着单链结构化DNA的解离是WC双链体形成的限速步骤。pH 7.0和pH 5.5时的表观活化能差异反映了pH显著影响G-四链体、I-基序和沃森-克里克双链体之间的结构竞争，这也暗示了I-基序在生物调节中可能具有生物学作用。
