G-quadruplexes are non-canonical secondary structures of DNA, which form under physiologically relevant conditions in guanine-rich sequences. There is significant evidence indicating that these structures may act as roadblocks for DNA replication, transcription, and repair. Blocking of these genomic maintenance processes by the induced stabilisation of such structures may have potential for the clinical treatment of genetic disorders. One question remains unanswered: what exactly do replication and repair proteins do once they encounter these DNA structures?
We aim to directly visualise individual replication proteins encountering G-quadruplexes in real time. We have developed a single-molecule fluorescence assay using DNA constructs with G-quadruplex structures. These DNA constructs can be attached to a microscope cover slip in a microfluidic flow cell. Upon introduction of DNA polymerases, the DNA construct will be replicated. We visualise this replication by monitoring the fluorescence intensity of a DNA stain. Changes in the fluorescence intensity provide a real-time insight into whether the polymerases (i) become stalled, or (ii) can bypass the structure. Our results show that a formed G-quadruplex efficiently blocks bacterial and eukaryotic DNA polymerases. We use fluorescently labelled polymerases to monitor their behaviour upon stalling. There is a current lack of understanding as to whether a stalled polymerase remains DNA-bound, dissociates, or undergoes rapid exchange with other polymerases.
Our single-molecule assay is the first-of-its-kind to explore how G-quadruplex structures impair replication proteins. This assay can be extended to catalogue the bypass capabilities of various replication proteins. Ultimately, these assays will give valuable insight into the mechanism of action of potential new therapeutics.