Saudi Cultural Missions Theses & Dissertations
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Item Restricted Structural basis for the roles of single and double Holliday Junctions formed from Human telomeric Nucleic Acids (HJTNA)(Univeristy College London, 2024-04-28) Alanazi, Abeer; Shozeb, HaiderBackground: 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 proteinDNA 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.13 0