Main, Kavit Harpal Singh; (2022) Targeting Twist: Single Molecule Insights into the Effect of DNA Supercoiling on Topoisomerase Interactions and Topoisomerase Inhibitor Chemotherapeutics. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Topoisomerase I (TOP1) is an essential enzyme in maintaining genomic stability in higher eukaryotes. Implicated in fundamental DNA transactions which remodel helical structure, TOP1 relieves excessive superhelical stress by catalysing the nicking, rotation, and religation of supercoiled DNA within a complex cellular environment. It is this necessity that has validated the ubiquitous TOP1 as a key target for anticancer therapeutics, namely by the prototypical camptothecin class of TOP1 inhibitors. Despite being used for decades, these therapeutics remain challenged by labile pharmacophores, the emergence of resistance, and off-target toxicity. With a comprehensive understanding of the mechanics of TOP1 inhibition remaining elusive, a complete description of this mechanism is necessary to drive the refinement and development of TOP1 inhibitors with improved selectivity, specificity, and safety. Underpinning this is a better understanding between the dynamics of the therapeutic target, TOP1, and its cellular substrate – supercoiled DNA. This study therefore employs a top-down approach in which clinical considerations are used to direct research at the nanoscale. Through use of Atomic Force Microscopy (AFM), the effect of negative superhelical stress on DNA structure is probed, revealing global compaction and partial relaxation driven by the onset of local helical defects. The effect of negative supercoiling on molecular recognition and enzymatic structural specificity is subsequently explored with triplex forming oligonucleotides (TFOs) and human TOP1 interaction – providing direct evidence of TOP1-TOP1 interactions and a secondary TOP1 binding site. These visual observations are complemented by optical tweezers experimentation which is also employed to characterise the kinetics of DNA religation by TOP1. The efficacy of an established camptothecin and novel indenoisoquinoline on increasing this religation timescale is clearly demonstrated on relaxed DNA and may be an important contributing factor in determining in vivo cytotoxicity across the highly heterogenous structural landscape of euchromatin.

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