Alanazi, Abeer;
(2024)
Structural basis for the roles of single and double Holliday Junctions formed from Human telomeric Nucleic Acids (HJTNA).
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
BACKGROUND: Telomeres, nucleoprotein complexes at chromosome ends, are crucial for genomic stability. Cancer cells maintain telomere length via telomerase or alternative lengthening of telomeres (ALT). The ALT pathway, observed in 15% of cancers, involves recombination and employs Holliday junction intermediates. These 4-way DNA motifs are dynamic structures influenced by cations, particularly Magnesium (Mg²⁺), leading to conformational changes. AIMS: This study aims to explore Holliday junction stability and mobility in the context of ALT, focusing on the impact of G-rich sequences. Leveraging single-molecule Förster resonance energy transfer (smFRET) and X-ray crystallography, we aim to understand the influence of varying Mg²⁺ ion concentrations on the Holliday junctions. Additionally, we investigate protein-Holliday junction complexes and assess the role of Mg²⁺ ions in protein-DNA binding affinity. METHODOLOGY: smFRET and X-ray crystallography have been employed to study Holliday junction structures. Microscale thermophoresis (MST) quantified protein-DNA binding affinity at different Mg²⁺ ion concentrations. T4 endonuclease VII, T7 endonuclease I, and the WRWYRGGRYWRW peptide were tested for their affinity under varying Mg²⁺ conditions. RESULTS AND CONCLUSIONS: The results highlight the direct influence of Mg²⁺ ions on Holliday junction stability. Protein-DNA binding affinity was observed through MST, with T4 endonuclease VII, T7 endonuclease I, and the WRWYRGGRYWRW peptide exhibiting persistent affinity at lower Mg²⁺ concentrations. However, affinity diminished at higher concentrations. smFRET analysis provided insights into branch migration rates across diverse Mg²⁺ ion conditions, suggesting a potential strategy for targeting ALT-positive cancer cells by stabilizing Holliday junction conformation. This study offers valuable insights into the ALT mechanism.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Structural basis for the roles of single and double Holliday Junctions formed from Human telomeric Nucleic Acids (HJTNA) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2024. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/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 > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10190180 |
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