Visualising protein induced changes in DNA conformation by Atomic Force Microscopy

The interactions between nucleic acids and proteins underpin gene regulation, fundamental cellular processes, and new bioengineering technologies. While sequencing-based methodologies offer insight into genome-wide protein binding, a comprehensive understanding of these processes goes beyond to include the structures and mechanics involved in these processes using biophysical techniques at the single-molecule level. By applying high-resolution Atomic Force Microscopy (AFM) to this challenge this thesis explores some of the structural and mechanical mechanisms underlying cellular interactions, to illuminate the impact of DNA structure and topology on DNA-protein binding.
Through optimisation of surface immobilisation protocols, high-resolution AFM imaging reveals DNA structures with resolution of the double helix and aberrations from the ideal structure, shedding light on how proteins interact with the structure and the conformational changes in nucleoprotein complexes. These methodologies, in combination with bulk biophysical approaches, are used to determine the previously unknown nuclear role of NDP52, revealing for the first time the monomer structure of the protein and a novel mode of dimerisation. Furthermore, AFM imaging provides information on the activity of NDP52 when complexed with DNA, visualising looped and bridged structures formed between NDP52 and DNA.
AFM was finally used to study the effect of supercoiling on the binding of Cas9 to DNA by imaging changes in the structure of supercoiled DNA substrates when bound to the dCas9 nucleoprotein. When bound by dCas9 the supercoiled substrate is relaxed, revealing the effect that R-loop formation within the protein has on the bound DNA. Relaxation of both on and off target minicircles indicate that, despite mismatches, R-loop formation is complete for off target sequences of this substrate. These findings show the opportunities offered by AFM to understand the role of DNA structure and conformation in molecular interactions.