Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.699943
Title: Mapping protein-DNA interactions using UV cross-linking and mass spectrometry
Author: Flett, Fiona Jane
ISNI:       0000 0004 5990 9673
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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Abstract:
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.
Supervisor: Interthal, Heidrun ; Harrington, Lea Sponsor: Biotechnology and Biological Sciences Research Council (BBSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.699943  DOI: Not available
Keywords: UV cross-linking ; mass spectrometry
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