Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands.
Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands.
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DOI：
10.1021/ja902281d
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发表时间：
2009-08-12
影响因子：
15
通讯作者：
Hurley LH
中科院分区：
文献类型：
--
作者：
Palumbo SL;Ebbinghaus SW;Hurley LH
hTERT核心启动子包含一个由12个连续的G-片段组成的富含G的区域，包含3个Sp1结合位点，并具有形成多个G-四链体的潜力。从鸟嘌呤束的12次运行中，选择9个推定的hTERT G-四链体形成序列，使用圆二色性和Taq聚合酶终止测定来测定G-四链体的形成和稳定性。从生物物理和化学分析的结果表明，总环的大小和结构稳定性之间的近似负相关。使用Taq聚合酶阻滞试验和硫酸二甲酯足迹法对全长hTERT G丰富序列的研究揭示了从该序列形成独特的端到端堆叠的G-四链体结构。这种结构由一个完全平行的G-四链体组成，由四个连续的G-束形成，连接到另一个非典型的G-四链体，由两对连续的G-束形成，由26个碱基的环分开。这个26个碱基的环可能形成稳定的发夹结构，这将解释这个G-四链体的意外稳定性。值得注意的是，这种串联的G-四链体结构在全长序列中的形成掩盖了所有三个Sp1结合位点，这被预测为产生hTERT启动子活性的显着抑制。此外，我们的研究表明，抑制端粒酶活性的一些G-四链体配体不仅是通过靶向端粒G-四链体，但也通过稳定的hTERT启动子G-四链体。
The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing three Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanine tracts, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase arrest assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes.
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影响因子：
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