Department of Biophysics, Johns Hopkins University, Baltimore, MD, United States.
Department of Biophysics, Johns Hopkins University, Baltimore, MD, United States; Physics Frontier Center (Center for Physics of Living Cells), University of Illinois, Urbana, IL, United States.
Methods Enzymol. 2022;672:283-297. doi: 10.1016/bs.mie.2022.03.065. Epub 2022 Apr 22.
The G-rich single-stranded telomere overhang can self-fold into G-quadruplex (G4) structure both in vivo and in vitro. In somatic cells, telomeres shorten progressively due to the end-replication. In stem cells, however, telomeres are replenished by a special enzyme, telomerase which synthesizes single-stranded telomere overhang. The active extension by the telomerase releases G-rich overhang segmentally in 5' to 3' direction as the overhang folds into G4 structure after successive elongation. To replicate such vectorial G4 folding process, we employed a superhelicase, Rep-X to release the G-rich sequence gradually. Using single-molecule assay we demonstrated that the folded conformation achieved by the vectorial folding is inherently different from the post-folding where the entire overhang is allowed to fold at once. In addition, the vectorially folded overhangs are less stable and more accessible to a complementary C-rich strand and the telomere binding protein, POT1 compared to the post-folded state. The higher accessibility may have implications for the facile loading of shelterin proteins after DNA replication.
富含 G 的单链端粒突出序列在体内和体外都可以自我折叠成 G-四链体 (G4) 结构。在体细胞中，端粒由于末端复制而逐渐缩短。然而，在干细胞中，端粒通过一种特殊的酶端粒酶来补充，端粒酶合成单链端粒突出序列。端粒酶的活性延伸会将富含 G 的突出序列分段从 5' 到 3' 方向释放，因为突出序列在连续延伸后折叠成 G4 结构。为了复制这种有向的 G4 折叠过程，我们使用了 Rep-X 超螺旋酶来逐渐释放富含 G 的序列。通过单分子检测，我们证明了通过有向折叠实现的折叠构象与折叠后允许整个突出序列一次性折叠的构象在本质上是不同的。此外，与折叠后状态相比，有向折叠的突出序列稳定性较低，更容易与互补的 C 丰富链和端粒结合蛋白 POT1 结合。更高的可及性可能对 DNA 复制后庇护蛋白的易于加载具有影响。
