Tracking and Tracing Complex DNA Structures and DNA-Protein Interactions Implicated in Aging and Disease

While the genetic code of DNA is undeniably fundamental for cellular development, proliferation and function, the significance of DNA structure has largely been overlooked. DNA structure is intrinsically linked to function, influencing processes such as DNA packaging into chromatin, unwinding of the double helix, protein binding and formation of alternative structures. All of which are critical for accurate DNA replication and gene expression, which can be implicated in human diseases.

Atomic Force Microscopy was used as a single-molecule imaging technique to probe the structure of complex DNA molecules, including knots and catenanes. With nanometre resolution, we have established a technique to observe which DNA strand is over-passing and which strand is under-passing, thereby explicitly determining the topology of DNA. The analysis of AFM images can be challenging due to high volume data sets. Therefore, an automated pipeline was developed to accurately classify the topology of these molecules.

Using this technique to observe changes to DNA structure, we looked at DNA-protein interaction. The Shelterin complex caps and protects telomeres from DNA damage proteins. When looking at the binding of Shelterin to telomeric repeat sequences, novel intra- and intermolecular bridging mechanisms were observed, mediated by the TRFH domain of TRF2. This mechanism provides insight into the protective roles of the Shelterin complex at telomeres.

Telomeres also form various alternative structures in the cell. They consist of the G-rich DNA repeat sequence TTAGGGn, prone to forming G-quadruplex structures (G4s). However, little is known about the formation of these structures in double-stranded telomeric DNA. Using AFM, the formation of G4s in double-stranded telomeric DNA was detected after physiological heating and the addition of potassium ions. On the addition of lithium ions, these structures appear to collapse. This information provides valuable insight into G4 formation and protection at telomeres.