Oxidised bases in DNA are removed by a number of DNA glycosylases in the first step of base excision repair. These include 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like 1 (NTH1) and the Nei-like proteins, NEIL1, NEIL2 and NEIL3. While NEIL1 and NEIL2 are relatively well characterized, the function of NEIL3 is still not fully understood. Although all three proteins show homology to the Escherichia coli Fpg/Nei family, NEIL3 is the largest member with an extended C-terminal domain and contains a valine instead of the highly conserved proline residue at amino acid position two. While it has been reported that recombinant murine NEIL3 shows DNA glycosylase and AP lyase activities in vitro, its biological role remains unclear. Therefore, to gain an insight into the function of NEIL3 in vivo, the full-length human NEIL3 cDNA has been expressed in Saccharomyces cerevisae as a prelude to undertake a yeast 2-hybrid screen to determine specific protein-protein interactions. To date, cDNA library screenings for potential hNEIL3 interactors have been completed and clones expressing potential interactors have been isolated, sequenced and analysed. This data, along with the results of other confirmatory experiments are presented in this thesis. Furthermore, clones of Pichia pastoris harbouring an expression cassette with full-length human NEIL3 or mouse NEIL1 or truncated versions of hNEIL3 with amino acid length 1-394 and 1-502 cDNA have been generated in preparation for its overexpression in a eukaryotic system. It is envisaged that the expression of 6XHis tagged hNEIL3 in P. pastoris will enable the purification of hNEIL3 protein that can be used in enzyme assays and for further in vitro investigations of putative protein interactions discovered by yeast two-hybrid (Y2H) screen.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577213 |
| Date | January 2013 |
| Creators | Roedl, T. |
| Publisher | University of Salford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://usir.salford.ac.uk/29368/ |
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