Trenaman, Anna Louise
Trypanosoma brucei BRCA2 in the regulation of genome stability and DNA repair.
PhD thesis, University of Glasgow.
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Trypanosoma brucei is a protistan parasite of mammals that evades its host’s immune responses by antigenic variation, which in T. brucei involves the periodic switching of the Variant Surface Glycoprotein (VSG) coat to antigenically distinct variants. The T. brucei genome contains a huge archive of silent VSG genes that are expressed from specialised expression sites, only one of which is actively transcribed at any one time. Copying of silent VSG genes into the active expression site has been shown to occur by homologous recombination, as mutation of the RAD51 recombinase and a distantly related gene, RAD51-3, impairs this process. BRCA2 is a protein that binds and regulates the function of Rad51 during homologous recombination. Mutation of BRCA2 in bloodstream form T. brucei leads to increased sensitivity to DNA damaging agents, and impairments in homologous recombination, RAD51 subnuclear foci formation and VSG switching, suggesting that it too acts in recombination-repair and antigenic variation. Beyond these phenotypes, an accumulation of putative gross chromosomal rearrangements in the megabase chromosomes of the T. brucei genome and a novel replication phenotype were also observed, and the basis of both these processes was unclear. T. brucei BRCA2 is highly unusual relative to orthologues in other eukaryotes, as the protein contains an expansion in the number of RAD51-binding BRC repeat motifs, which are arranged in a tandem repeat array that has not been observed elsewhere.
In order to examine the function of BRCA2 in the maintenance of genome stability in T. brucei, brca2 homozygous mutants were generated in procyclic form TREU 927 and Lister 427 cells. Analysis of genomic stability by Southern blotting and pulsed field agarose gel electrophoresis revealed that BRCA2’s function in the maintenance of genome stability appears to be either bloodstream form-specific, or plays a more substantial role in this life cycle stage. To examine the function of the BRC repeat expansion, cell lines containing variants of BRCA2 with reduced numbers of BRC repeats were generated, expressed in brca2 homozygous mutants and phenotype analysis carried out. Growth and DNA repair were restored by the expression of virtually all variants, suggesting the BRC repeat expansion is not an adaptation for general genome maintenance, though the repair activity of a variant with a single BRC repeat appeared to differ between bloodstream and procyclic form parasites. In contrast to this, a striking correlation between BRC repeat number and the regulation of RAD51 subnuclear dynamics was observed, showing that the BRC array expansion has important functional significance.
GST pull-down analysis was used to examine the domains of T. brucei BRCA2 that interact with RAD51, revealing an extent of interaction not apparent in BRCA2 orthologues in other organisms. This complexity of interaction was further analysed by immunolocalisation of BRCA2 and RAD51, before and after DNA damage, which showed potentially dynamic co-localisation of the two repair factors. Finally, a putative interaction between T. brucei BRCA2 and CDC45 was tested both in vitro and in vivo, but could not be validated, suggesting it does not provide an explanation for the replication defects observed in bloodstream form brca2-/- mutant cells. All of the analyses above shed light on the function of the BRCA2 protein in the regulation of homologous recombination in T. brucei.
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