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Item Restricted Investigation the mechanism behind radiotesistance in SPRTN depleted cells(University of Oxford, 2025) Aljarbou, Ftoon Ali; Ramadan, Kristijan; RUGGIANO, AnnamariaSPRTN is a DNA-dependent metalloprotease essential for the replication-coupled removal of DNA–protein crosslinks (DPCs), thereby maintaining genome integrity. Loss of SPRTN function leads to the accumulation of covalent DNA–protein adducts and is associated with altered chromatin states. While SPRTN-deficient cells are hypersensitive to classical crosslinking agents such as camptothecin, cisplatin, and other chemotherapeutics, we unexpectedly observed that they exhibit increased resistance to ionising radiation (IR). This paradox prompted us to investigate the underlying chromatin-based mechanisms that might explain their radioresistance. In Chapter 3, we evaluated methods for DPC quantification to establish a robust platform for studying DPC dynamics. Using formaldehyde-treated wild-type cells, we compared the RADAR and STAR assays. The RADAR protocol, which employs guanidine thiocyanate to inactivate nucleases and preserve labile crosslinks, detected DPCs in a clear dose- and time-dependent manner. In contrast, the STAR assay, which lacks chaotropic agents, failed to capture the same DPC increases likely due to proteolytic degradation of DPCs during lysis. Based on these findings, RADAR was adopted for all subsequent DPC burden assessments. SPRTN-deficient cells exhibit impaired DNA damage signalling, with neutral comet assays showing more residual double-strand breaks (DSBs) after ionizing radiation (IR) compared to wild-type cells. Despite this increased damage, γH2AX and 53BP1 foci were less intense and fewer in ΔSPRTN cells, and checkpoint activation was reduced, as indicated by lower phosphorylated CHK1 levels post-radiation. Furthermore, SPRTN deficiency induces a shift toward a repressive chromatin state, even under basal conditions. Disrupting heterochromatin, via HDAC inhibitors (panobinostat, TSA) or knockdown of heterochromatin-associated factors like H1.2, HP1, and SUV39H1, restored radiosensitivity and DDR signalling. This suggests chromatin accessibility directly modulates DDR. Formaldehyde treatment in wild-type cells, which increased DPCs and resistance to IR, mimicked the ΔSPRTN phenotype, indicating DPC accumulation may alter chromatin configuration. Chromatin compaction hinders DDR execution by restricting repair protein recruitment, allowing ΔSPRTN cells to evade apoptosis and continue proliferating. These findings highlight the role of DPCs in shielding DNA lesions, reducing DDR activation. This has translational implications for cancers with defective DPC repair, like Ruijs-Aalfs syndrome, which may possess epigenetically compacted chromatin and intrinsic radioresistance. Therapeutically, targeting heterochromatin formation could resensitize these cells to IR. HDAC inhibition reversed radioresistance in ΔSPRTN cells. Future studies on diverse cell types and in vivo models are needed, with high- LET radiation potentially bypassing chromatin shielding. In conclusion, our results demonstrate that SPRTN loss elicits a protective chromatin condensation that attenuates DDR signalling and suppresses apoptosis in cancer cell lines examined, particularly HeLa. This work uncovers a previously unrecognised link between DPC accumulation, chromatin architecture, and DDR efficiency, and sets the foundation for chromatin-targeted radiosensitisation strategies in DPC repair-deficient cancers. Additional experiments would further clarify this finding, supporting deeper investigation into chromatin-targeted therapies and strengthens the case for SPRTN's role in mediating radioresistance.9 0Item 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.14 0Item Restricted Taxonomy and Diversification of Cyclamen L. Using Next-Generation Sequencing Approaches(University of Reading, 2023-10-27) Hubini, Ahmed; Culham, AlastairThere are 24 species of Cyclamen, which found predominantly in the Mediterranean region and its surrounding areas in Europe, Asia, and North Africa. Over the years, scientists have studied the genus morphologically, cytologically, and more recent, systematically to unravel the mystery surrounding its evolutionary past. Using Next-Generation Sequencing (NGS) techniques, we assembled complete chloroplast, nuclear ribosomal DNA, and 20 mitochondrial coding genes using multiple assemblers. Comparative analysis revealed that the chloroplast genomes in Cyclamen were largely similar to those of other angiosperms, with C. somalense being a unique case, displaying the loss of four ndh genes. Notably, ycf15 emerged as a pseudogene across the genus, similar to certain Primulaceae family members, suggesting its potential as a molecular marker for future investigations. Phylogenetic trees constructed based on the assembled genetic data displayed strong congruence, except for certain species that showed variations in their placement within the trees. The phylogenetic trees in this thesis showed the Cyclamen is monophyletic in the family Primulaceae. The mitochondrial-based tree was well-supported but remained unresolved, particularly in the Cilicium group, where a polytomy was observed. However, based on the trees, the results suggest that C. alpinum should belong to the Cilicium group instead of Coum. Additionally, C. africanum should be treated as a subspecies of hederifolium rather than a separate species. Furthermore, network approach was used to investigate the relationship in the Hederifolium group collected from Greece with different ploidy levels. Based on complete chloroplast sequences there are 17 distinct haplotypes and ten haplotypes based on nuclear ribosomal DNA. The results demonstrate a poor correlation between ploidy level and locations and current C. hederifolium subspecies classifications, due to limited genetic variation. The agreement between this thesis and the previous findings provides further support for the complex cytological characteristics exhibited by Cyclamen species within this group.11 0Item Restricted DNA REPAIR IN THE SULFOLOBUS ARCHEON(Saudi Digital Library, 2023-11-19) Qalyoubi, Azoof; Bray, SianObjective: The purpose of this study was to identify putative operons in the Sulfolobus archaean genus and explore the role of its genes in DNA repair mechanisms. By doing so, we aim to contribute to the understanding of the complex processes involved in DNA repair in these organisms and to conduct a comparative study of the operons containing Mre11, Rad50 (SbcC, SbcD), HerA, and NurA across the archaea and bacteria domains by studying the genes occurring alongside these four primary genes in the operons across different species. Methods: To search for putative operons, we used a combination of bioinformatics tools (Terminal, Python scripts and Gephi program) and experimental techniques (MicrobesOnline software, Blast, NCBI). We first conducted a genome-wide search for genes involved in DNA repair and then analysed their genomic context to identify potential operons. Results: Our analysis identified several putative operons involved in DNA repair in Sulfolobus and bacteria species. These operons are highly conserved across different strains of Sulfolobus and bacteria and are involved in a variety of DNA repair mechanisms, including base excision repair, nucleotide excision repair, and homologous recombination. Our experiments also confirmed the function of these operons in DNA repair and provided insights into their regulation and interaction with other repair pathways. Conclusion: Our study provides a comprehensive overview of DNA repair mechanisms in the Sulfolobus archaean genus and many bacterial species and sheds light on the role of putative operons in these processes. Our findings have important implications for the field of bioinformatics and genetic research as they provide a foundation for further studies on DNA repair mechanisms in Sulfolobus and other organisms. Keywords: DNA repair, Sulfolobus, operons, bioinformatics, genetic research, Mre11, Rad50, NurA, HerA, bacteria species, thermophilic, mesophilic, putative operon, MicrobesOnline24 0