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Through the use of a biochemical technique that allows single strands of DNA to be visualized, the dynamics of nucleotide excision repair proteins was studied at the single molecule level. This DNA tightrope technique has allowed for the interaction of proteins with double stranded, single stranded, crowded and damaged DNA to be observed. Bacterial nucleotide excision repair proteins UvrC and UvrBC were loaded onto undamaged DNA to understand how UvrC locates the UvrB-DNA pre-incision complex. It was obseNed that UvrC facilitates UvrB binding to DNA in the absence of UvrA and as this UvrBC complex was abundant, indicates that UvrC is most likely present in an UvrBC complex in vivo. Mutant studies demonstrated that UvrBC binds to DNA through UvrB's DNA binding domain, signifying that the UvrBC complex forms in order to help UvrC locate an UvrB pre-incision DNA damage complex and to stop unchecked endonuclease activity. It was proposed that this UvrBC complex may also benefit UvrC in maneuvering around obstructions on the DNA. Within a cell DNA will always be involved in a multitude of differentreactions concurrently, therefore obseNations on how NER proteins behaved on crowded DNA was studied. These results demonstrated that both UvrBC and UvrC have difficulties overcoming protein obstructions. This suggests that the main mechanism of movement of UvrC and UvrBC on the DNA is a one dimensional search, with the possible capability of switching to a 3D search. The eukaryotic NER protein XPD was also imaged, demonstrating a single stranded DNA binding preference over that of double stranded DNA. The XPD protein was also obseNed to have an absolute requirement for A TP in order to have motion on the DNA. Once damage was introduced into the DNA it was obseNed that UvrB facilitates UvrC binding to damaged DNA, through the UvrB-DNA binding domain. This previously unreported damage detection mechanism of UvrBC originates from UvrB's ~-hairpin motif's interaction with DNA. It was therefore suggested that UvrBC's damage .detection is a redundancy system in the NER pathway.

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Retroelements are some of the most successful parasites studied because of their ability to reverse transcribe and permanently integrate into the host genome. Host cells have, therefore, evolved multiple control mechanisms, such as cellular restriction factors, to protect their genomes from the pathogenic and mutagenic effects of retroelements. Identification of the full complement of these proteins is vital to comprehend the capacity of the host to regulate these genetic parasites. Human MOV10 is a putative RNA helicase with inhibitory or stimulatory roles in the replication of several RNA viruses, and the homologs of which play vital roles in the restriction of viruses and endogenous retroelements. Furthermore, MOV10 interacts with antiviral APOBEC3 proteins and core post-transcriptional RNA silencing machinery, all of which colocalise in cytoplasmic mRNA processing bodies and stress granules. Considering MOV10 cellular associations and homolog functions, the capacity of MOV10 to regulate the replication of a diverse panel of genetically distinct retroelements was investigated here. Ectopically overexpressed MOV10 potently restricts the replication of retroviruses as well as the propagation of LTR and non-LTR endogenous retroelements. Significantly, RNAi-mediated silencing of endogenous MOV10 enhances the replication of endogenous retroelements, but not exogenous retroviruses demonstrating that natural levels of MOV10 suppress retrotransposition. MOV10 overexpression decreases the level of HIV-1 genomic RNA packaged into nascent virions and also impacts the accumulation of reverse transcription products in target cells. The molecular mechanism/s by which MOV10 inhibits retroelements remains unclear, however, the anti-retroelement activities of MOV10 and APOBEC3 proteins are independent. Moreover, MOV10 is not essential for miRNA-mediated translation repression or slicer activity in cultured cells. In sum, ectopically overexpressed human MOV10 inhibits divergent exogenous and endogenous retroelements and, more significantly, the capacity of endogenous MOV10 to specifically suppress retrotransposition highlights it as a potential restriction factor of human retrotransposons in somatic cells.

Mitochondria supply the majority of cellular ATP and have additional important roles in calcium signalling, apoptosis and lipid metabolism. Mutations in or loss of mitochondrial DNA (mtDNA) can cause neurodegeneration and has been linked to Parkinson‟s disease. However, the pathological consequences of mtDNA loss in neurons are very poorly understood. We have used the fruitfly, Drosophila melanogaster, to study how loss of mtDNA affects neuronal function. We find that both ubiquitous RNAi and overexpression of the mtDNA binding protein TFAM causes reduced mtDNA content and lethality. TFAM RNAi or overexpression specifically in motor neurons causes locomotion defects during development and age-related behavioural defects in adult flies. This demonstrates that maintenance of mtDNA is required for normal motor neuron function. In further behavioural assays we show reductions in evoked jump response, demonstrating that mtDNA loss inhibits motor neuron activity. Also, using confocal imaging we find that mtDNA loss does not cause neuronal loss or changes in synaptic bouton number during development. However, we observe a significant decrease in the number of mitochondria and the number of pre-synaptic active zones (the sites of neurotransmitter release) in motor neurons with reduced mtDNA content. Taken together these data show that neuronal mtDNA loss results in defects in synaptic development and reduced motor neuron function. We further show that these phenotypes can also result from other methods of mtDNA depletion, such as mitochondrial specific expression of the restriction enzyme XhoI. Furthermore, possible roles of TOR pathway and autophagy on motoneuron synapses were investigated. The phenotypes observed may represent the initial pathological consequences of mtDNA loss in neurodegenerative disease.

DNA 5- Cytosine methylation is a modification that occurs in mammalian cells predominantly at CpG sequences, through addition of a methyl group to the C5 position of cytosine. It is a commonly held view in the current field of epigenetics that the aberrant methylation patterns observed in gene promoter regions are associated with transcriptional repression in the absence of genetic mutations. This epigenetic phenomenon is frequently reported in malignant haematopoiesis, where genes involved in cell growth and differentiation programmes are shown to be transcriptionally silenced leading to unchecked cellular proliferation. Although strongly implicated in gene shut down, it is still unclear whether promoter region methylation triggers gene repression, or if DNA methylation arises as a consequence of transcriptional gene silencing via some other mechanism. This is in part because no-one has yet managed to dissect in vivo the precise sequence of the events associated with the appearance of aberrant methylation patterns at the promoter region of actively transcribing genes. This thesis documents a research programme aiming to exogenously impose de novo cytosine methylation to specific regions of a relevant endogenous promoter in order to study spatial and temporal interactions between targeted DNA methylation and other components of the epigenetic regulatory network. The gene selected for this study was CDKN2B, a cell cycle regulating tumour suppressor gene strongly implicated in Myelodysplastic Syndrome - MDS and Acute Myeloid Leukaemia - AML. Site-biased zinc finger: DNA methyltransferase fusion proteins were specifically designed with the aim of depositing low and high density de novo methylation at different regions of the CDKN2B promoter, in an attempt to simulate methylation patterns associated with disease and to thus determine their effect on gene expression. We demonstrate here for the first time the establishment of low density de novo methylation at this endogenous locus, characterized by highly specific target site methylation downstream of zinc finger binding sites. Examination of the distribution pattern and the spread of methylation from the seeded methylation hotspots suggest that 5’ CpG positions in flanking sequences remain unmethylated, whilst 3’CpG acquire de novo methylation marks derived from the action of endogenous DNA methyltransferases. The effects of methylation spread over longer time scales on the formation of broader and more uniform methylation patterns are currently being investigated. Our data also shows that once established, the methylation pattern is inherited through successive cell divisions. Gene expression analysis indicates sustained transcriptional activity of the low density methylated promoter despite the presence of these de novo methyl marks. Overall, our results demonstrate that targeted low density methylation to the promoter region of the CDKN2B gene can be established and maintained epigenetically through subsequent cell generations, but has no effect on the transcriptional regulation of the gene. To date we have not been successful in targeting high density methylation to the same region to comprehensively evaluate the bilateral relationship between a densely methylated promoter and its transcriptional activity/regulation. The contribution of the transcription factor VEZF1 in mediating methylation protection at the CDKN2B promoter was also examined, and we showed for the first time that this transcription factor is strongly associated with this promoter in vivo. This may have important implications in our understanding of CDKN2B expression and regulation in the disease process. Related studies also revealed that TET proteins may play a significant role in cell cycle entry, which was another hitherto unknown phenomenon. In conclusion, we feel that the work in this thesis has advanced the field of targeted methylation, the further implementation of which will certainly further our understanding of key epigenetic mechanisms such as the cellular response to de novo methylation and its role in the regulation of an important cell cycle and disease associated gene.

Zinc is an essential micronutrient indispensable for all forms of life, and disturbances in its homeostasis predispose the body to zinc imbalance-related diseases in man and animals. Mechanisms exist that enable organisms to regulate this metal element, which include proteins that control zinc entry into the cell, chelation whilst in the cell, and also extrusion out of the cell. ZnT1 (SLC30A1) is a zinc exporter originally cloned in mammalian cells but its homologue has also been identified in piscine species. In the present study, we have used genetics and molecular approaches in a model vertebrate system (the zebrafish Danio rerio) to elucidate the nutritional and functional importance of zinc and its transporters in the regulation of some crucial biological processes in the body. The Znt1 mutant of zebrafish strain sa0014 was created by the Sanger Centre using TILLING technology. The mutant fish carries a premature stop codon in the znt1 (slc30a1) gene resulting in a protein that is forty (40) amino acids shorter than the wild- type. A colony of this strain was generated and the mutation studied for its effects on ability of embryos to regulate Zn2+ ions needed for biological processes, such as growth and development, gene expression, zinc acquisition and extrusion, and also its effects on extracellular-regulated kinases (ERK1/2). Investigations were also conducted on embryos with znt1 gene knockdown, through deployment of antisense morpholino- modified oligonucleotides (MO) in embryos of wild-type background, and in all of the studies it was shown that zebrafish embryos with Znt1 deficiencies display subtle but interesting phenotype, often having disturbances in zinc regulation and cell signalling when compared to their wild-type counterparts. Functional studies in adult fish revealed that znt1 mutation affects zinc absorption and transporter regulation as well as diurnal breeding pattern, which was plausibly linked to dysregulation of circadian rhythm- controlled genes and ERK signalling. In addition, investigation was also conducted on the function of Zip10 in embryos and it was found that the zip10 gene is essential in early development through its involvement in epithelial-mesenchymal transition (EMT) and cell migration. This gene was also shown to be important in hatching of zebrafish embryos through its involvement in the proper development and functioning of hatching gland tissue. Interestingly, knockdown of the zip10 gene by MO reduced the expression of hatching gland markers as well as the abundance of Zn2+ ions in the hatching gland cells (HGC) resulting in delayed hatching whereas znt1 knockdown produced the opposite effects. The relevance of all these studies in health and diseases of man and animals are discussed.

Within all cells, complex molecular systems exist that are responsible for maintaining genome stability by detecting and repairing dangerous alterations in DNA. Ensuring the accurate and efficient functioning of such systems is necessary for the preservation of DNA integrity and avoidance of disease. The flexible and diverse modes of DNA-binding exhibited by human p53 permits this ‘guardian of the genome’ to elicit versatile cellular activities that are crucial in monitoring threats to genome dynamics and conducting appropriate responses. In conjunction with its sequence-specific DNA-binding activity that is essential to target gene transactivation, p53 can bind to unusual DNA structures independent of DNA sequence and it has been proposed this activity may allow p53 to interact with detrimental secondary structures that arise in unstable genomic regions. To provide further insight into p53-DNA interactions, an in vitro DNA binding assay was developed that was used to characterise binding properties towards several DNA molecules to allow comparison of non-specific, sequence-specific and structurespecific binding. It was determined that unusual structures in DNA significantly enhanced p53 binding in non-sequence specific DNA and that the presence of internal hairpin regions induced binding comparable to sequence-specific binding. In vivo p53-DNA interactions were also quantified using chromatin immunoprecipitation and variations in preference to different response element sequences was ascertained. DNA binding is also central to the ability of Ku proteins to function as essential components of non-homologous end joining and telomere maintenance in eukaryotes. Prokaryotic homologues of Ku proteins that function as homodimers in two-component repair systems have also been identified. Recently, 3 Ku homologues in Streptomyces coelicolor were reported, but very little is currently known regarding their biological activity. It was discovered that all 3 Ku proteins exhibited varied independent DNA-binding properties that were influenced by DNA topology, size and end-structure. Unusually for Ku, it was found 1 of these proteins exhibited strong binding to single-stranded DNA. Precipitation assays determined that these proteins may act as DNA end synapsis mediators during the DNA endjoining process and ligation experiments revealed Ku was responsible for rigidifying DNAs or completely inhibiting ligation activity, probably via DNA end-protection activity. Experimental evidence indicated that specific interactions could occur between S. coelicolor Ku suggesting these proteins form both homodimers and heterodimers.

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