Departments of Physics and Biological Sciences, Kent State University, Kent, OH 44242.
Proc Natl Acad Sci U S A. 2014 Feb 25;111(8):2990-5. doi: 10.1073/pnas.1321436111. Epub 2014 Feb 10.
Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.
端粒的 3' 端带有单链的 G 突出端，该端可被复制蛋白 A（RPA）识别为 DNA 损伤部位。端粒保护蛋白 1（POT1）/POT1 相互作用蛋白 1（TPP1）异二聚体特异性结合于单链端粒 DNA（ssTEL），保护 G 突出端免受 RPA 结合。G 突出端会自发折叠成各种 G-四链体（GQ）构象。目前尚不清楚 GQ 形成是否会影响 POT1/TPP1 与 RPA 竞争结合 ssTEL 的能力。我们通过单分子Förster 共振能量转移实验表明，POT1 可稳定地加载到邻近折叠 GQ 的最小 DNA 序列上。在 150mM K(+) 条件下，POT1 加载会使反平行 GQ 解折叠，而平行构象仍然保持折叠状态。POT1/TPP1 的加载可阻止 RPA 以两个数量级的水平结合折叠和未折叠的端粒。在 150mM Na(+) 条件下，ssTEL 仅形成不太稳定的反平行 GQ，因此观察不到这种保护作用。这些结果表明，端粒突出端的 GQ 形成可能有助于抑制 DNA 损伤信号。
