Mechanisms behind Protein-DNA Interactions Unveiled with Molecular Simulation and Atomic Force Microscopy

DNA specificity underlines the fundamental basis through which many different proteins perform their myriad roles within organisms, a key example being the genome structuring performed by the nucleoid-associated proteins (NAPs). This thesis presents results found through the usage of all-atom molecular dynamics (MD) and in-liquid atomic force microscopy (AFM) towards understanding DNA-protein interactions for a variety of these systems. One of the most abundant NAPs is the histone-like protein from E. coli strain U93 (HU), which specifically binds to sites of DNA damage and creates sharp bends to aid in DNA repair. Here, simulations reveal how the protein diffuses along and between strands of DNA towards finding a binding site. Once a site of damage is found, we show a clear multimodality in the binding of HU to DNA, observed in both MD and AFM. AFM imaging also shows aggregation of DNA by HU, which simulations show to be highly energetically favourable, explaining how HU condenses the nucleoid, and why it is key to biofilm stability. A pair of evolutionarily related proteins, ParB and Noc, have been used to study how specificity evolved in order to allow new regulatory functions to be fulfilled. The use of MD here explained the roles of key mutations unable to be sampled in experiments, and in-silico mutagenesis was applied to map out all potential pathways from changing amino acids and nucleotides. Beyond the specific recognition of DNA by proteins, a mechanism through which proteins can be mechanically encapsulated by a DNA origami structure has also been studied. A new methodology to simulate such structures has been developed, and then applied to understand the interactions of GFP with the side of a structure, revealing strong, non-specific interactions between the protein and the DNA that would allow the protein to remain caught within a DNA box.