Many DNA repair pathways have been documented in Trypanosoma brucei but less attention has been paid to damage tolerance, a reaction in which lesion bypass is needed, in particular to ensure continued genome replication. Such bypass is promoted by translesion DNA polymerases (TLS Pols). T. brucei has ~15 TLS polymerases candidate genes, only two of which have been functionally examined to date. Understanding the roles provided by TLS Pols could reveal new aspects of T. brucei biology. Here, I examine the activities of TLS Pol Nu (PolN), TbPolZ and TbPolQ (HelQ) in bloodstream cells. RNAi against TbPolN results in slowed growth after ~24 hours, which is associated with altered DNA content, changed cell morphology and sensitivity to DNA damage. Surprisingly, growth and morphology defects are reduced after ~48 hours, without apparent RNAi reversion. In addition, depletion of the protein seems to lead to an aberrant distribution of the chromosomes, as visualised by telomere fluorescent in situ hybridization. TbPolN epitope tagging demonstrates a discrete localisation of the protein at the periphery of the nucleus in the absence of damage, with a more widespread, but non-uniform localisation after damage. EdU labelling and γH2A analysis after TbPolN knockdown reveal a decrease in proliferating cells, which accumulate nuclear DNA damage. Finally, we show that TbPolN interacts with a nuclear putative non-canonical PolyA polymerase. Taken together, these data suggest TbPolN may be involved in T. brucei nuclear DNA maintenance. RNAi of TbPolZ (zeta) did not impair growth but resulted in increased sensitivity to methyl methanesulphonate (MMS) damage and UV radiation, suggesting a possible role in the response against both genotoxic agents. Generation of TbPolZ null mutants confirmed that the protein is non-essential and plays a role in genotoxic damage repair. Surprisingly, TbPolZ epitope tagging not only showed a nuclear signal, but a mitochondrial signal was also detected. These data were supported by immunoprecipitation, where mitochondrial proteins were obtained as potential interaction partners. These data suggest a contribution of TbPolZ to both nuclear and kinetoplast genome maintenance. Targeted RNAi of the third putative TLS-related factor, TbHelQ, was unsuccessful. Despite this, sequence analysis of the protein indicates that its current annotation as a PolQ homologue is inaccurate, since the predicted protein is not a joint polymerase-helicase like in other eukaryotes, but only a putative helicase. Hence, it is suggested it should be renamed TbHelQ. Immunoprecipitation and colocalisation analyses indicate a possible role of TbHelQ in homologous recombination, given the potential interaction of the factor with BRCA2 and other factors involved in this repair process. Notably, the predicted interactome of TbHelQ differs from that of TbPolN, suggesting discrete functions in T. brucei. Taken together, these data reveal widespread and variant functions of three putative TLS DNA polymerases in the parasite genome biology, suggesting a possible role in the maintenance of genome integrity in T. brucei.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739225 |
| Date | January 2017 |
| Creators | Zurita Leal, Andrea Cristina |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/8897/ |
Page generated in 0.0021 seconds