Haynes, Philip John;
(2025)
Biophysical Studies of DNA-Protein-Small Molecule Interactions.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
DNA takes on various forms as it complexes with other biomolecules, folds, and adopts a range of topologies. G-quadruplex DNA (G4-DNA) is one such form and has been proposed to play regulatory roles in oncogene expression, DNA repair, transcription, replication and telomere maintenance. To study G4-DNA under conditions that better mimic its in vivo environment, including topologically constrained flanking DNA, we have developed assays using DNA minicircles. Through Förster Resonance Energy Transfer (FRET) based kinetic assays, we have demonstrated how the presence of flanking bases elevates the stability of G4-DNA and, in some cases, hinders or enhances the stabilising effect of small molecules. To study the impact that backbone-ligation, and therefore increased constraint, have on DNA minicircle structure, we used Atomic Force Microscopy (AFM). Further to DNA topology, DNA-protein complexes are at the centre to both how DNA is organised and how cells respond to DNA damage. The DNA damage response pathway conserves genomic stability and is maintained by a tight network of DNA-protein interactions. Poly (ADP-ribose) polymerase-1 (PARP1) is a strand-break repair protein that is clinically targeted in cancer cells that are deficient in one or more DNA repair pathways. However, the role that PARP1 plays in shaping DNA architecture remains elusive. Through the application of single-molecular biophysical tools, we have discovered and characterised PARP1-induced compaction of DNA at physiologically relevant concentrations. AFM was deployed to characterise the typical structures of DNA-PARP1 complexes, the degree of DNA-bound PARP1 polymerisation, the influence of PARP1 inhibitors on DNA-PARP1(2) affinity, and the extent to which PARP1 binds to damaged versus undamaged DNA. To this end, we have gathered evidence for an alternative functionality of PARP1, which may provide a novel mode-of-action for PARP1 inhibitors.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Biophysical Studies of DNA-Protein-Small Molecule Interactions |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10204667 |
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