Use this URL to cite or link to this record in EThOS:
Title: A putative photolyase from Haloferax volcanii
Author: Wu, Winnie
ISNI:       0000 0004 2684 3639
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2009
Availability of Full Text:
Access from EThOS:
Access from Institution:
Extreme halophilic archaea, like Haloferax volcanii, which thrive in hypersaline environments, are subject to intense UV irradiation in their natural habitats; however, the mechanisms underlying their superb tolerance to UV-induced cytotoxic DNA lesions, mainly cyclobutane pyrimidine dimers (Pyr<>Pyr) and pyrimidine – pyrimidone (6-4) photoproducts (Pyr [6-4] pyr), remain unclear. Among the various DNA repair pathways used by halophilic archaea, photoreactivation is the simplest and most efficient reversal mechanism. In this light-dependent process, the monomeric enzyme, photolyase, repairs the DNA lesion and causes the splitting of the cyclobutane ring via the absorption of visible light. This mechanism has been observed in several species of halophilic archaea, where light repair results in higher cell survival than dark repair. Annotation of the complete genome sequence of the UV-tolerant haloarcheon, Halobacterium sp. NRC-1 has identified 2 photolyase-like genes, phr 1 and phr 2 where knock-out studies have confirmed the function of the encoded Phr2 protein as a CPD photolyase. However, this protein has not been expressed and functionally characterized from any halophilic archaeon known to date. With this in mind, a project was designed to investigate the expression and function of the phr encoded proteins from Haloferax volcanii with the aim to elucidate their function in DNA repair. Haloferax volcanii is a halophilic archaeon that grows optimally at 42 degree C under pH7, and requires an optimum salt concentration between 1.5M -2.5M. Using the known phr gene sequences from Halobacterium sp. NRC-1, two putative gene homologues, phr1 and phr2, were identified from the unannotated Hfx. volcanii genome. Homology modelling to known photolyase structures of Escherichia coli and Anacystis nidulans was conducted where the hypothetical structure of the protein closely resembled a deavaflavin type of photolyase containing the 8-HDF chromophore. The putative genes were amplified and transformed into the native strain to observe an over-expression of the Phr proteins. Due to the low copy number of the vector used, protein expression was too low to be detected; this led to a modification scheme for the vector to include a polyhistidine tag upstream of the gene for Nickel affinity purification. This strategy resulted in the purification of a native protein, pitA, a reflection of the low expression of Phr2 in the native strain. Heterologous expression in E. coli hosts resulted in the production of inclusion bodies that required renaturation to re-gain the protein native conformation. The Phr2 protein was purified, solubilised using 8M urea and refolded using various tested refolding buffers. Circular dichroism spectroscopy revealed that the refolded protein has secondary structures with a high ratio of Î[±]-helixes. A novel in-vivo assay developed for detecting the UV-sensitivity of the native overexpression strain strongly confirmed a photoreactivating function of the phr2-encoded protein but not for the phr1-encoded gene product. A cell viability assay indicated that white light illumination resulted in higher cell numbers for the Phr2 strain post UV irradiation. Lastly, an immunoassay was used for the detection of photoreactivating activity of the re-folded protein and the native over-expression strain. Results indicated that while the re-folded protein did not exhibit repair, the native over-expression strain once again showed a white light enhancement of repair of dimers. These results and findings are discussed in context with the current literature on photoreactivation and DNA repair in other halophilic archaea.
Supervisor: Hough, David ; Hejmadi, Momna Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available