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Title: An investigation into the initiation of VSG switching in Trypanosoma brucei
Author: Devlin, Rebecca
ISNI:       0000 0004 5373 0103
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2015
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Trypanosoma brucei, the eukaryotic parasite that causes human African trypanosomiasis in humans, evades the immune system through antigenic variation. T. brucei antigenic variation involves the periodic switching of the variant surface glycoprotein (VSG) coat to an antigenically distinct variant. A single VSG is expressed on the cell surface at any one time, but the T. brucei genome contains a vast number of silent VSGs. To be expressed, a VSG must be located in a specialised VSG blood stream form expression site (VSG BES). Silent VSGs are copied into VSG BES by homologous recombination. Several proteins have been demonstrated to be involved in this process but how VSG switching is initiated remains unclear. Four putative DNA repair factors were identified in T. brucei, whose eukaryotic homologues play a range of roles in DNA repair and other aspects of genome maintenance. These were two RecQ-like helicases, a Mus81 endonuclease and a Pif1 family helicase (PIF6). To examine whether these factors play a role in DNA repair and VSG switching, mutants were generated in blood stream form T. brucei cells. Analysis of RecQ1 by RNAi knockdown revealed it to be an essential gene in bloodstream form T. brucei, possibly involved in nuclear DNA replication. Phenotypic analysis of recq2 mutants suggests that RECQ2 is involved in the repair of a range of DNA damaging agents. Furthermore, analysis of survival following DSB induction suggests RECQ2 is involved in the repair of DNA DSBs, including those in the active VSG BES. VSG switching analysis showed that recq2-/- mutants have an elevated VSG switching rate and increase in recombination events upstream of the active VSG. These analyses suggest that RECQ2 suppresses VSG switching in T. brucei by suppressing recombination events near the active VSG. Analysis of mus81 mutants showed mus81-/- mutants to be sensitive to agents inducing replication stalling and DNA breaks, and that MUS81 is important in the repair of DSBs. PIF6 appears to be a complicated DNA repair factor, different from MUS81 and RECQ2. pif6+/- and pif6-/- mutants appear to be more resistant to MMS than wild type cells, though more sensitive to the replication stalling agent hydroxyurea. pif6 mutants do not appear to be more sensitive to DSBs than wild type cells and may even be more resistant. It is unclear whether PIF6 is involved in VSG switching and more work is required on this factor to attempt to understand its DNA repair and VSG switching function in T. brucei. These analyses shed light on the DNA repair functions of four previously uncharacterised T. brucei proteins. In particular, observations that RECQ2 is deficient in repairing DSBs upstream of the active VSG and mutants exhibit an elevated VSG switching rate cannot be reconciled with current thinking that direct formation of DSBs in this location initiates VSG switching. This suggests that the initiation of VSG switching is more complex than currently thought and requires careful further study and consideration of the relevance of using direct DSBs in this location to model VSG switching.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General) ; QH301 Biology ; QH426 Genetics ; QR Microbiology