The supercoiling of DNA refers to the over-winding or under-winding of the double helix, which occurs due to torsional stress. Supercoiled DNA can form a plait-like structure of inter-wound superhelicies referred to as plectonemes. The supercoiling of DNA affects biological processes that occur on DNA, including DNA replication, transcription, and repair. Despite this involvement, in vitro studies on these processes largely work with relaxed DNA in the absence of supercoiling. This means that the physiological relevance of these findings is often limited, with the potential role of supercoiling in these replisomal processes being overlooked.
We have constructed linear 18-kb DNA substrates that contain six biotinylated nucleotides at each end. These substrates can be tethered to a streptavidin-functionalised coverslip through biotin/streptavidin binding at multiple sites. These multiple bonds allow for the template to become topologically constrained, as they prevent the DNA strands from rotating around themselves to alleviate imposed stress. The intercalating dye, SYTOX orange, can then be used to induce supercoiling within this topologically constrained template. Using single-molecule TIRF microscopy, we can visualise individual DNA molecules and determine the position of plectonemes, which appear as bright fluorescent spots. In the absence of template modifications and enzymes, plectonemes diffuse along the DNA. However, in the presence of modifications or bound enzymes, the plectonemes remain stationary at the site of the modification. These findings reveal a relationship between DNA topology and the activity of DNA modifying enzymes. This work will provide insight into how replisomal processes manage complex topology, or, potentially, how this complex topology is facilitating these replisomal processes.