Kusi-Appauh Nicholas, Mueller Stefan H, Ralph Stephen F, Yurieva Olga, O'Donnell Michael E, Lewis Jacob S, Spenkelink Lisanne M
Molecular Horizons and School of Science, University of Wollongong.
Howard Hughes Medical Institute, Rockefeller University; Laboratory of DNA Replication, Rockefeller University.
J Vis Exp. 2025 Apr 4(218). doi: 10.3791/68080.
The ability of proteins involved in eukaryotic DNA replication to overcome obstacles - such as protein and DNA 'roadblocks' - is critical for ensuring faithful genome duplication. G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase δ, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.
参与真核生物DNA复制的蛋白质克服诸如蛋白质和DNA“路障”等障碍的能力对于确保基因组的准确复制至关重要。G-四链体是在DNA富含鸟嘌呤的区域形成的高阶核酸结构，已被证明可作为障碍，干扰基因组维持途径。本研究引入了一种基于实时荧光显微镜的方法，以观察DNA聚合酶与G-四链体结构的相互作用。含有G-四链体的短引物DNA寡核苷酸固定在微流控流动池内功能化的玻璃盖玻片上。引入荧光标记的DNA聚合酶，以便随时间监测其行为和化学计量。这种方法能够观察到聚合酶被G-四链体阻碍时的行为。具体而言，使用荧光标记的酵母聚合酶δ发现，遇到G-四链体时，聚合酶会经历一个持续的结合和解离循环。这种单分子检测方法可用于研究各种DNA维持蛋白与DNA底物上的障碍之间的相互作用。
