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Title: Mismatch repair, recombination and genetic variability in Trypanosoma brucei
Author: Barnes, Rebecca Louise
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2006
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The protozoan parasite Trypanosoma brucei has a complex life cycle with stages in mammalian hosts, where it is the causative agent of sleeping sickness in humans and nagana in cattle, and in the tsetse fly vector. In order to evade the host immune system, T. brucei undergoes a process called antigenic variation in the mammalian bloodstream. In this process, a single Variant Surface Glycoprotein (VSG) is expressed on the cell surface, acting as a protective coat. The molecular identity of the VSG coat is periodically and spontaneously changed by a number of different switching mechanisms. T. brucei is known to have conserved DNA repair pathways, including homologous recombination (HR) and mismatch repair (MMR). While the central recombination factor RAD51 and its paralogue RAD51-3 have been shown to be important, but not essential, in VSG switching, a number of other factors, including the MMR proteins MSH2 and MLH1, seem not to be involved. Work in this thesis sought to examine several aspects of MMR function in T. brucei, and concentrated on homologues of the bacterial MutS protein. The requirements for substrate length and sequence homology in T. brucei HR were studied using a DNA transformation assay. It was shown that reduction in either the length or the sequence identity of recombination substrates causes a significant reduction in the transformation efficiency of linear DNA, at least at an interstitial site. Genetic disruption of the MSH2 gene only seemed to affect HR using substrates over 100 bp in length and with 5% divergence from the target sequence; shorter sequences and sequences with either 0% or 11% mismatches apparently remained unaffected. A number of transformants from all classes of transformation retained an undisturbed copy of the target locus, hypothesised to be due to low-level trisomy within the population. In addition, and at a very low rate, distinct recombination events, resulting in observable changes in the T. brucei chromosomes, were observed. This work reveals some of the factors which influence the pathways of recombination used by T. brucei. A potential role for T. brucei homologues of the meiosis-specific MutS homologues MSH4 and MSH5 was also examined. Sequence comparisons show that these genes are present in T. brucei and the related kinetoplastids, T. cruzi and L. major. Like their orthologues in other organisms, T. brucei MSH4 and MSH5 lack a detectably functional mismatch interaction domain. Although MSH4 and MSH5 would only be expected to be required at the epimastigote life cycle stage, expression of MSH5 can be detected by northern blot in procyclic form and bloodstream stage cells. Although creating genetic knockouts of these genes was not successful, attempts were made to force expression of MSH4 and MSH5 ORFs from an ectopic locus, though this did not disrupt MMR function, nor reveal other observable phenotypes. Finally, potential variation in MMR gene sequence and MMR functions in different T. brucei strains and subspecies was investigated. Many bacterial strains, known as mutators, have mutations in MMR genes, causing impaired MMR function and therefore increased variability in the population. It has recently been reported that this phenomenon is also observed in T. cruzi. MSH2 and RAD51 nucleotide and protein sequences were compared between nine T. brucei strains, and showed extremely low levels of polymorphism. However, four T. brucei strains were found to vary in their tolerance to the DNA damaging agents MNNG, H2O2 and MMS; whether this is due to differences in MMR, another DNA repair pathway, or drug uptake, is yet to be determined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available