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Helicases and DNA dependent ATPases of Sulfolobus solfataricus /Richards, Jodi Dominique. January 2008 (has links)
Thesis (Ph.D.) - University of St Andrews, May 2008.
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Investigation of the transcriptional response of Sulfolobus solfataricus to damaging agents /Munro, Stacey. January 2009 (has links)
Thesis (Ph.D.) - University of St Andrews, April 2009.
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Investigation of the transcriptional response of Sulfolobus solfataricus to damaging agentsMunro, Stacey January 2009 (has links)
It is vital for the survival of an organism that it can repair damage to its DNA. Exogenous and endogenous sources of damage are dealt with by a variety of repair pathways that have evolved to repair specific types of damage. Organisms in the archaeal domain, the third domain of life, contain homologues of many of the eukaryotic repair proteins, however little is known about how damage is detected in the archaeal domain. Microarray studies in the archaeal species Sulfolobus solfataricus determined a number of genes whose expression was effected by UV radiation (work by Dr D Götz). The change in expression of nine of these genes was confirmed by RT real time PCR. The expression of these genes was then investigated after exposure to different damaging agents, Mitomycin C, Methyl methane sulfonate, Phleomycin and Hydrogen peroxide. The expression of two genes, transcription factor tfb-3 and cell division control gene cdc6-2, was up regulated in all damage conditions. There was a huge induction of the dps-like gene (sso2079) after hydrogen peroxide damage. Transcription from this genes promoter was shown to be strong in vitro (work by Dr S Paytubi) suggesting a repressor was controlling the gene in vivo. A palindromic repeat in the promoter of the dps-like gene was used to ‘fish’ for a transcriptional repressor and the Sso2273 protein, a homologue of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheria, was identified as a possible repressor. Sso2273 was expressed and purified, and its crystal structure solved, its paralogue, Sso0669, was also expressed and purified. Electrophoretic mobility shift assays showed that the Sso2273 protein does not bind DNA, and had no effect on transcription from any promoter used in in vitro transcription assays. However Sso0669 appeared to inhibit transcription, although the inhibition was not sequence specific. A knockout strain of S. solfataricus PBL2025 missing the sso2273 gene was produced and used in microarray experiments in an attempt to determine the role of Sso2273 within the cell. The absence of Sso2273 appeared to have no effect on the expression of the dps-like gene, however strong repression of an operon containing genes involved in Sulphur assimilation was observed.
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Splitting, joining and cutting : mechanistic studies of enzymes that manipulate DNAMcRobbie, Anne-Marie M. January 2010 (has links)
DNA is a reactive and dynamic molecule that is continually damaged by both exogenous and endogenous agents. Various DNA repair pathways have evolved to ensure the faithful replication of the genome. One such pathway, nucleotide excision repair (NER), involves the concerted action of several proteins to repair helix-distorting lesions that arise following exposure to UV light. Mutation of NER proteins is associated with several genetic diseases, including xeroderma pigmentosum that can arise upon mutation of the DNA helicase, XPD. The consequences of introducing human mutations into the gene encoding XPD from Sulfolobus acidocaldarius (SacXPD) were investigated to shed light on the molecular basis of XPD-related diseases. XPD is a 5’-3’ DNA helicase that requires an iron-sulphur (FeS) cluster for activity (Rudolf et al., 2006). Several proteins related to SacXPD, including human XPD, human FancJ and E. coli DinG, also rely on an FeS cluster for DNA unwinding (Rudolf et al., 2006; Pugh et al., 2008; Ren et al., 2009). Sequence analysis of the homologous protein, DinG, from Staphylococcus aureus (SarDinG) suggests that this protein does not encode a FeS cluster. In addition, SarDinG comprises an N-terminal extension with homology to the epsilon domain of polymerase III from E. coli. This thesis describes the purification and characterisation of SarDinG. During replication, DNA lesions or other ‘roadblocks’, such as DNA-bound proteins, can lead to replication fork stalling or collapse. To maintain genomic integrity, the fork must be restored and replication restarted. In archaea, the DNA helicase Hel308 is thought to play a role in this process by removing the lagging strands of stalled forks, thereby promoting fork repair by homologous recombination. Potential roles of Hel308 during replication fork repair are discussed in this thesis. The mechanism by which Hel308 moves along and unwinds DNA was also investigated using a combined structural and biophysical approach. The exchange of DNA between homologous strands, catalysed by a RecA family protein (RecA in bacteria, RAD51 in eukaryotes, and RadA in archaea), defines homologous recombination. While bacteria encode a single RecA protein, both eukaryotes and archaea encode multiple paralogues that have implications in the regulation of RAD51 and RadA activity, respectively. This thesis describes the purification and characterisation of one of the RadA paralogues (Sso2452) in archaea.
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