Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes.

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.