Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, 100 Technology Drive, Pittsburgh, PA 15219, USA, Department of Physics, North Carolina State University, 2401 Stinson Drive, Raleigh, NC, 27695, USA, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA and Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Nucleic Acids Res. 2013 Dec;41(22):10323-33. doi: 10.1093/nar/gkt813. Epub 2013 Sep 13.
Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.
先前的证据表明，端粒类似于常见的脆性位点，给 DNA 复制带来了挑战。端粒 TTAGGG 重复序列处复制叉推进的确切障碍尚不清楚，但据推测包括富含 G 链上的 G-四链体 (G4)。在这里，我们研究了复制性 DNA 聚合酶 δ/增殖细胞核抗原/复制因子 C 复合物在模拟前导 C 丰富和滞后 G 丰富链的端粒模板上的 DNA 合成和推进。与富含 C 的模板和非端粒模板相比，富含 G 的模板上聚合酶停滞增加。用 Li(+)代替 K(+)作为阳离子，或使用含有 7-脱氮-G 残基的模板来抑制 G4 形成，并没有减轻 G 残基内 Pol δ 暂停部位。此外，我们提供的证据表明，与线性寡核苷酸相比，在末端受约束的单链环状 TTAGGG 模板上，G4 折叠的稳定性较低。用 G4 配体 BRACO-19 人为稳定环状模板上的 G4 结构会抑制 Pol δ 进入富含 G 的重复序列。酵母和人类 Pol δ 复合物也得到了类似的结果。我们的数据表明，G4 形成不是端粒滞后链上聚合酶停滞所必需的，并且除了稳定的 G4 之外，还存在另一种机制导致端粒处的复制停滞。
