Targeting Twist: Single Molecule Insights into the Topological Dependence of DNA - Topoisomerase Interactions

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Topoisomerase inhibitors are a vital class of therapeutics used in oncology. They target ubiquitous topoisomerase enzymes which are responsible for maintaining the state of cellular DNA. Topoisomerase-targeting therapeutics are currently challenged by off-target toxicity and the emergence of drug resistance, and a comprehensive understanding of their mechanism of action remains elusive. Furthermore, some topoisomerases remain untargeted despite a strong rational for their effectiveness in eliciting cell death. A complete description of the catalytic cycle of topoisomerases and the mechanics of their inhibition will aid in improving the selectivity, specificity, and safety of these therapeutics and facilitate an improved drug refinement and development pipeline.Underpinning this is a better understanding of the dynamics between the therapeutic target, topoisomerases, and cellular DNA. In particular, a consideration of how these enzymes interact with DNA under topological or supercoiling stress, has limited exploration.Topoisomerases relieve stress in DNA by cutting and rearranging DNA through nanometre conformational changes on the order of seconds within a complex cellular environment. Here, we perform single molecule in vitro studies to improve our understanding of how DNA structure varies under topological stress, and how this affects topoisomerase activity, using Atomic Force Microscopy. Atomic Force Microscopy scans a sharp tip over molecules immobilised on a surface in fluid, to 'feel' the contours of the molecule with nanometre precision and sub-second temporal resolution.The overarching aim of my project is to therefore study the topological dependence of DNAtopoisomerase/ topoisomerase inhibitors interactions to facilitate therapeutic refinement.We use small closed circular DNA molecules with controlled levels of superhelical stress to mimic the globally underwound state of genomic eukaryotic DNA and investigate changes in higher order structure in response to supercoiling. To this end, we have observed the onsetof defects in the double helical structure of closed circular DNA at physiological levels of superhelical stress. These defects increase the local flexibility of DNA resulting in increased conformational heterogeneity. Currently, we are using bacterial topoisomerases, in particularGyrase, to gain preliminary results about binding affinities and preferences of these enzymes to supercoiled DNA. We observe preferential binding of Gyrase, with the majority of binding events located at the outside of both open and kinked conformers inducing conformationalchanges in DNA proximal to the binding site. These observations provide insight into the spatial and energetic requirements of these enzymes, upon which we are able to optimise our imaging conditions further.Subsequently, we aim to optimise a protocol that allows us to achieve high-resolution dynamic imaging of eukaryotic topoisomerases (TOP1 and TOP2) to supercoiled DNA. This will allow us to gain information regarding topoisomerase structure but also the conformational changes that the enzymes undergo during catalysis. This will be followed by the addition of gold-standard topoisomerase inhibitors; the selection of which will be based on current practice guidelines such that our research is driven by clinical practice. In turn, these experiments will be complemented by the use of novel topoisomerase inhibitors, such as with the idenoisoquinolone drug class, to visually compare mechanistic inhibitory effects.