Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.689422
Title: Structural and functional analysis of the XPF/Mus81 family of endonucleases
Author: Fadden, A. J.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2006
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Abstract:
This thesis describes structural and functional analysis of members of the XPF/Mus81 family of junction-specific endonucleases. These enzymes recognise and nick double-stranded DNA within a variety of flaps, bubbles and branched DNA substrates. Genetic and biochemical evidence indicates that these endonucleases have important functions in nucleotide excision repair, DNA interstrand crosslink repair, recombination and replication fork repair, and thereby help maintain genomic integrity. Eukaryotic XPF orthologues are large multidomain proteins that are not suitable for structural studies at present. For this reason, an XPF orthologue from the crenarchaeote Aeropyrum pernix has been used to shed light on the mechanism of XPF action. The crystal structure of the core nuclease domain of A. pernix XPF has been solved to 2.1 A resolution. This together with other structures detailed in the literature has provided insight into enzyme architecture and possible mechanisms of substrate binding and cleavage. Attempts to co-crystallise A. pernix XPF with short oligonucleotides akin to repair substrates are detailed. Other evidence in the literature has implicated the processivity factor PCNA in stimulating crenarchaeal XPF activity. With this in mind, recombinant heterotrimeric A. pernix PCNA has been expressed and been complexed with various A. pernix XPF constructs. The repair helicase XPB has also been added to form a ternary repair complex. Structural studies of these complexes by crystallography and electron microscopy have been initiated and some crystals have been produced. Bioinformatic analysis of eukaryotic Mus81 orthologues has identified a region of conserved sequence of previously unknown structure or function. This putative domain appears to be conserved in most eukaryotic Mus8,l orthologues. The human domain was overexpressed for biochemical and structural analyses. Deletion of this domain from within Mus81 appears to affect endonuclease activity against splayed arm substrates in vitro, suggesting it has a role in protein-DNA interaction. Crystals of the human domain have been produced, although diffraction data sufficient for structure determination has not been obtained.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.689422  DOI: Not available
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