Characterisation of factors that regulate homologous recombination and antigenic variation in Trypanosoma brucei

QH301 Biology;SF600 Veterinary Medicine;QH426 Genetics

Hartley, Claire Louise ; McCulloch, Richard

University:University of Glasgow

Department:School of Veterinary Medicine

关键词: Trypanosomes, homologous recombination, antigenic variation, BRCA2, RAD51, genomic instability;   

Others  :  http://theses.gla.ac.uk/410/1/2008hartleyphd.pdf

来源: University of Glasgow

【 摘 要 】

Trypanosoma brucei is an evolutionarily divergent eukaryotic parasite of mammals in sub-Saharan Africa and is transmitted by the tsetse fly vector. To evade the mammalian immune response, T. brucei utilises antigenic variation, which involves switches in the Variant Surface Glycoprotein (VSG) expressed on the cell surface. Such reactions can occur at very high rates (~10-3 switches/cell/generation) and occur primarily by the recombination of VSG genes, selected from an enormous silent archive, into specialised expression sites. It has been previously shown that such VSG switching is a form of homologous recombination, as mutation of RAD51 and a related gene, RAD51-3, impairs the process. BRCA2 has emerged as a significant regulatory factor during RAD51-catalysed recombination. In humans, BRCA2 contains eight BRC repeats, six of which have been shown to bind RAD51. Similar repeats are present in BRCA2 from other organisms, though normally in smaller numbers. This thesis describes a T. brucei BRCA2 homologue that appears exceptional in that it contains up to 12 BRC repeats. Furthermore, the sequence degeneracy that is observed between the BRC repeats in most organisms is absent in T. brucei, with all but the C-terminal proximal repeat being identical. It was hypothesised that this unusual BRCA2 organisation is due to the high levels of RAD51-directed recombination needed during antigenic variation. To examine the function of the putative T. brucei BRCA2 homologue, mutants were generated and found to display impaired growth, sensitivity to induced DNA damage, impairment in the ability to form sub-nuclear RAD51 foci, a reduced ability to recombine DNA constructs into their genome and a reduction in frequency of VSG switching, all of which are consistent with roles for BRCA2 in DNA repair and recombination. Furthermore, genome instability in the mutants was observed through the loss of silent VSG gene copies and substantial reductions in the size of the mega-base chromosomes. Interestingly, other chromosome classes (the so-called mini- and intermediate-chromosomes) appear not to be susceptible to such instability. A potentially novel function for BRCA2 was identified through DNA content analysis of the T. brucei BRCA2 mutants. Mutation of BRCA2 was shown to result in an accumulation of cells with aberrant DNA content that is most readily explained by an increased number of cells that undergo cytokinesis without having completed nuclear division, phenotypes that are not observed in other T. brucei recombination mutants, such as RAD51. This result suggests that BRCA2 has a role in the regulation of cell division, with mutation causing impaired replication of T. brucei nuclear DNA, but without a cell cycle stall, leading to the accumulation of chromosomal aberrations. In order to investigate the potential role of T. brucei BRCA2 in DNA replication and the unusual BRC repeat organisation phenotypes further, various truncations of BRCA2 were expressed in a mutant background. Cell lines expressing BRCA2 with only 1 BRC repeat displayed reduced efficiency in recombination, DNA repair and RAD51 foci formation, indicating that the large BRC repeat expansion in T. brucei BRCA2 plays a critical role in the proteins function. Expression of a BRCA2 variant encompassing only the region of the protein, C-terminal to the BRC repeats appeared able to function, at least partially, in regulating cell cycle progression. Moreover, this DNA replication role appears not to be provided by conserved DNA binding motifs present within the C terminus of BRCA2 since a fusion of T. brucei BRCA2 and the parasites homologue of the replication protein A 70 kDa subunit was impaired in cell division, but was proficient in repair of DNA damage. Taken together, these data infer that T. brucei BRCA2 possesses a function that is distinct from BRCA2’s role as a regulator of RAD51, and acts in DNA replication or cell division. In addition to the above research on BRCA2, I sought to examine the factors that interact with RAD51 in T. brucei. This work demonstrated that it is possible to add an epitope tag for tandem affinity purification (TAP) to the N-terminus of RAD51 in both the bloodstream and procyclic stages of T. brucei without disrupting its function. Preliminary data suggest that TAP is potentially a feasible way of examining RAD51 interacting factors.