Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.

中文翻译：

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多个 hPOT1-TPP1 协同展开从 3' 到 5' 的连续端粒 G-四链体，这是由于 3' 端结合偏好和端粒 DNA 结构的特征

端粒是与shelterin蛋白相关的DNA重复序列，并保护真核染色体的末端。人类端粒由 5'TTAGGG 重复序列组成，并以 3' 单链尾端（称为 G 突出端）结束，该尾端可与Shelterin 蛋白 hPOT1（人类端粒保护 1）特异性结合。体外研究表明，端粒 G 链可以折叠成稳定的连续 G 四链体 (G4)。在本研究中，我们研究了 hPOT1 如何与它的外壳蛋白伙伴 TPP1 复合，在钾溶液中与结构为连续 G4 的端粒序列结合。我们观察到多个 hPOT1-TPP1 的结合优先从 3' 向 5' 进行。我们从两个因素来解释这种方向性：(i) hPOT1-TPP1 对位于端粒序列 3' 端的结合位点的偏好和 (ii) hPOT1-TPP1 在钾中显示的协同结合。通过比较 K+ 和 Li+ 中的结合，我们证明了这种协同行为并非源于蛋白质-蛋白质相互作用，而是源于端粒 DNA 底物在钾中的连续 G4 结构。我们的研究表明，在生理条件下，POT1-TPP1 可能优先覆盖从 3' 端开始并朝向 5' 的端粒 G 突出端。