Mismatch repair in DNA recombination and antigenic variation in Trypanosoma brucei

Bell, Joanna Sharlene (2002) Mismatch repair in DNA recombination and antigenic variation in Trypanosoma brucei. PhD thesis, University of Glasgow.

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Abstract

African trypanosomes evade the mammalian host immune system for prolonged periods by periodically switching the protective Variant Surface Glycoprotein (VSG) coat expressed on their cell surface. Recombination reactions, which introduce novel VSG gene copies into specialised expression sites, constitute the principal mechanism of this antigenic variation process. Since VSG genes occupy cassettes with common upstream and downstream sequences it is hypothesised that VSG switching reactions occur primarily by homologous recombination. It has previously been shown that at least some of the VSG switching reactions are either catalysed or regulated by RAD51, a fundamental component of the homologous recombination machinery in eukaryotes. In order to investigate further the factors regulating antigenic variation we have characterised several components of the highly conserved post-replicative mismatch repair (MMR) system in T. brucei. The nuclear MMR system recognises mispaired bases in heteroduplex recombination intermediates preventing recombination between divergent DNA molecules, and thereby confining such reactions to highly homologous sequences. In yeast and mammals this system is composed of three homologues of the bacterial MutS enzyme and three bacterial MutL-related enzymes. We have identified five single-copy genes putatively encoding three MutS homologues and two MutL homologues, suggesting that most of the components of eukaryotic MMR are conserved in trypanosomes. Knockout mutants have been generated in two of the genes, MSH2 and MLH1, to investigate the activities of MMR in this organism. Both MSH2-deficient and MLH1- deficient trypanosomes exhibit microsatellite instability, a mutator phenotype, and tolerance to the methylating agent MNNG, demonstrating that these genes encode components of an active MMR system in T. brucei. Moreover, deletion of MSH2 increases the frequency of recombination between 3% divergent sequences, revealing that MMR is also involved in the regulation of recombination in this organism. Despite this, no effect on the frequency or mechanisms used in antigenic variation was observed in either MSH2 or MLH1 mutants, suggesting that MMR has little or no influence on this process, and that perhaps the homologous recombination reactions underlying antigenic variation proceed by a mechanism distinct from general recombination.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Richard McCulloch
Keywords: Biochemistry
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-71963
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 13:38
Last Modified: 17 May 2019 13:38
URI: http://theses.gla.ac.uk/id/eprint/71963

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