Protein-nucleic acid interactions play essential roles in all living cells in various cellular functions. The study of these interactions can reveal important structural and functional information. UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterisation of the specific sites of cross-linking is a challenging task. In this investigation, novel tools and methods have been developed for the investigation of DNA-protein interactions using UV cross-linking and mass spectrometry. These tools were developed towards their application for the characterisation of the complex between the eukaryotic DNA repair protein Tyrosyl-DNA phosphodiesterase 1 (Tdp1) and its DNA substrates. DNA-Tdp1 UV cross-linking was optimised using purified recombinant human Tdp1 and radioactively labelled DNA oligonucleotides containing UV photoactivatable 4- thio-thymidine or 5-iodouracil. Tdp1-DNA heteroconjugates were detected by SDS PAGE and Phosphorimaging. In order to analyse the DNA-Tdp1 heteroconjugates by mass spectrometry, they must first be enriched and hydrolysed by a protease and a nuclease. Here, a novel sample preparation protocol was developed for the enrichment of Tdp1 oligonucleotide-peptide heteroconjugates. Detection and analysis of oligonucleotide-peptide heteroconjugates using mass spectrometry is a challenging task. As a tool to optimise the various parameters involved, a synthetic DNA oligonucleotide-peptide heteroconjugate was constructed using click chemistry. RP-HPLC/ESI-FT-ICR-MS on a Bruker 12T SolariX in conjunction with CID fragmentation was used to unambiguously identify the site of the cross-link. Lastly, a novel 18O labeling approach was introduced to facilitate the identification of DNA-protein cross-links. This approach was shown to be suitable for the labeling of heteroconjugate species by testing it with the click heteroconjugate.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:699943 |
| Date | January 2014 |
| Creators | Flett, Fiona Jane |
| Contributors | Interthal, Heidrun ; Harrington, Lea |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17996 |
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