Unravelling the Invisible Complexities of the Genome

Synopsis

DNA in cells is highly dynamic - processes such as transcription, replication and protein binding events mechanically remodel DNA through bending, torsional strain, unwinding and structural transitions. Despite its importance, it is this dynamic nature and conformational complexity that makes explicitly determining these DNA mechanics challenging. Technological developments in high-resolution Atomic Force Microscopy (AFM) now enable us to routinely visualise single DNA molecules with sub-molecular resolution. However, AFM is still not routinely used to help solve problems inaccessible to the traditional tools of structural biology, due in part to a lack of software for quantitative structural characterisation. We have developed a single-molecule pipeline combining high-resolution AFM and quantitative image analysis methodologies to identify and quantify complex DNA structure and mechanics with nanometre resolution.

We use this pipeline to determine the mechanisms of DNA damage by anticancer metallodrugs based on platinum and copper, identifying differential effects such as single- and double-strand breaks in the DNA helix, in addition to compaction and aggregation effects. Beyond DNA damage, we can also assess the effect of complex DNA structures on transcription. We determine that single nick sites can drive up a large increase in R-loop formation, which present in a range of conformations, depending on defects in the template strand. Finally, we extend our pipeline to identify and quantify the volume and location of proteins bound to DNA to understand the dynamics and catalytic activity of topoisomerase II.