Jones, Alex and McKean, Paul and Benson, Fiona (2017) Characterisation of potential Holliday junction resolvases in Trypanosoma brucei. Masters thesis, Lancaster University.
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Abstract
Antigenic variation on the cell surface of the T. brucei is essential for the survival of the parasite in the host immune system. Finding mechanisms to stop variant surface glycoprotein (VSG) switching, and hence the parasites ability to evade the host immune system provides a promising opportunity to treat the infection. Holliday junctions are an essential part of homologous recombination, which is used for DNA repair and for the crossover of DNA. Identifying T. brucei proteins which act as Holliday junction resolvases are potential candidate targets for inhibiting VSG switching. In this project, bioinformatics methods were used to identify proteins with homology to human GEN1, a known Holliday junction resolvase. Purification of homologous proteins were carried out from the expression of bacterial cell lines. Parasite cell lines were created for the investigation of the homologous protein localisation and a BioID analysis to interrogate protein-protein interactions. Bioinformatics analyses identified two proteins with homology to human GEN1, TbFEN1 and TbRAD2. Of these two proteins, TbFEN1 underwent large scale purification and was used for the production of polyclonal antisera, where TbRAD2 was found to require stringent denaturing conditions for purification. Both proteins were seen to localise within the nucleus of the procyclic parasite. Finally, BioID analysis showed protein-protein interactions with TbFEN1 were found within the nucleus and a complex banding pattern of proteins observed. This work has been able to identify two proteins which potentially act as Holliday junction resolvases in the T. brucei. Further investigation of their function and protein interactions will be important to understand whether these provide functional targets for VSG switching.