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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:552399 |
| Date | January 2010 |
| Creators | McRobbie, Anne-Marie M. |
| Contributors | White, Malcolm F. |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/951 |
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