The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

中文翻译：

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端粒 G 突出端二级结构折叠动力学在酿酒酵母端粒维持调节中的作用。


真核染色体的末端通常含有富含 G 的 3' ssDNA 突出（G 突出）。必须保护该突出端免受核酸酶和 DNA 损伤反应机制的有害活动，并参与端粒酶（一种维持端粒完整性的核糖核蛋白复合物）的调节。这些功能由 DNA 结合蛋白（例如酿酒酵母中的 Cdc13）以及富含 G 的序列形成各种非 B DNA 结构的倾向介导。使用 CD 和 NMR 光谱，我们在此表明​​，酿酒酵母的 G 突出端根据其长度形成不同的基于 Hoogsteen 配对的二级结构。短端粒寡核苷酸形成 G-发夹，而它们的较长端粒寡核苷酸则形成平行和/或反平行 G-四链体 (G4)。无论其拓扑如何，非 B DNA 结构在体外表现出与 Cdc13 的结合受损，如电泳迁移率变动测定所示。重要的是，虽然 G4 结构形成相对较快，但 G 发夹折叠极其缓慢，表明大多数细胞周期中常见的短 G 突出端主要以单链寡核苷酸形式存在，是 Cdc13 的合适底物。使用 ChIP，我们发现 G4 结构的出现在 S 期晚期达到峰值，因此与长 G 突出端的积累相关。我们提出了一个模型，说明染色体末端非 B DNA 结构的时间和长度依赖性形成如何参与端粒维持。