Shanxi Key Laboratory of Aging Mechanism Research and Translational Applications, Center for Healthy Aging, Central Laboratory, Changzhi Medical College, Changzhi, Shanxi 046000, People's Republic of China.
State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.
Proc Natl Acad Sci U S A. 2024 Oct 29;121(44):e2401099121. doi: 10.1073/pnas.2401099121. Epub 2024 Oct 23.
Guanine-rich DNA forms G-quadruplexes (G4s) that play a critical role in essential cellular processes. Previous studies have mostly focused on intramolecular G4s composed of four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has not been strictly confirmed in the genome of living eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and two other independent G4-specific detections reveal that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of potential G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s readily form and even dominate at G4 sites that are theoretically capable of forming the intramolecular DNA G4s (dG4s) by themselves. Compared to dG4s, hG4s exhibit broader kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing it to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also establishes a sensitive and currently the only method for detecting the structural form of G4s in living cells.
富含鸟嘌呤的 DNA 形成 G-四链体 (G4s)，在重要的细胞过程中发挥关键作用。以前的研究主要集中在由单链上四个连续的鸟嘌呤链段 (G-链段) 组成的分子内 G4s 上。然而，这种结构形式在活真核细胞的基因组中尚未得到严格证实。在这里，我们报告了在活酵母细胞的基因组中形成由 DNA 和 RNA 的 G-链段组成的杂交 G4s (hG4s)。对冈崎片段合成的分析和另外两种独立的 G4 特异性检测表明，由于 RNA 的 G-链段的参与，hG4s 可以仅通过单个 DNA 鸟嘌呤-鸟嘌呤 (GG) 链段有效地形成。这一发现使酵母基因组中潜在的 G4 形成位点数量从 38 个增加到 587,694 个，增加了 15000 多倍。有趣的是，hG4s 很容易形成，甚至在理论上能够自身形成分子内 DNA G4s (dG4s) 的 G4 位点上占据主导地位。与 dG4s 相比，hG4s 表现出更广泛的动力学、更高的普遍性以及更大的结构多样性和稳定性。最重要的是，hG4 的形成通过 RNA 的参与与转录紧密偶联，使其能够以依赖转录的方式发挥作用。总的来说，我们的研究确立了 hG4s 是酵母基因组中占绝对优势的 G4 物种，并强调了对 G4s 在真核基因组中的结构形式、形成机制、普遍性和功能作用的重新认识。它还建立了一种敏感的、目前唯一用于检测活细胞中 G4 结构形式的方法。
