evades the host’s immune system and persists in the body through a process known as antigenic variation, which is the stochastic change of
s variant surface glycoprotein (VSG), and relies on homologous recombination. One of >2000
genes is expressed from a bloodstream expression site located in an inherently fragile subtelomeric region, with the
adjacent to a telomere. The key enzyme responsible for telomeric maintenance is telomerase reverse transcriptase (TbTERT), which extends the telomere by 6-12 bp per division (1,2). Without telomerase activity (in
mutants), the telomere loses 3-6 bp per generation (3), leading to short telomeres that have been linked to increased VSG switching (4). However, whether TbTERT influences repair pathway choice or recombination events driving VSG switching remains unexplored.
Mutants with disrupted
TERT were generated and a controlled double-strand break (DSB) was induced by the I-SceI endonuclease in the active expression site to examine the DNA damage response. We measured the length of the telomere using Southern blot and compared the parental and
tert⁻/⁻ mutants before induction of a break, to ensure that any differences in response were not due to initial telomere length variation. We saw that the initial telomere length was similar between the cell lines. Using VSGseq, we analysed
VSGs used for switching 7 days post-break in
tert⁻/⁻ mutants. We observed a noticeable, 3-fold increase in number of
VSGs used compared to parental cells. These results suggest that TERT may suppress access to the VSG archive during switching, while its deletion leads to hyper-recombinogenic state. Notably, this occurs independently of the early DNA damage response, as DNA resection and γH2A accumulation remained intact in
tert-/- cells. We are now tagging known factors such as Rad51-3 and PPL2 using TurboID, which will be followed by quantitative mass spectrometry analysis to identify proteins that accumulate at the site of the DSB. It will allow us to determine whether DNA repair chromatin composition differs between
tert-/- and parental cells, particularly when comparing subtelomeric and chromosome internal DSBs.
References
1. Bernards A, Michels PAM, Lincke CR, Borst P. Growth of chromosome ends in multiplying trypanosomes. Nature. 1983 Jun;303(5918):592–7. doi:10.1038/303592a0
2. Pays E, Laurent M, Delinte K, Van Meirvenne N, Steinert M. Differential size variations between transcriptionally active and inactive telomeres of Trypanosoma brucei. Nucleic Acids Res. 1983;11(23):8137–47. doi:10.1093/nar/11.23.8137
3. Dreesen O. Telomere structure and shortening in telomerase-deficient Trypanosoma brucei. Nucleic Acids Res. 2005 Aug 2;33(14):4536–43. doi:10.1093/nar/gki769
4. Hovel-Miner GA, Boothroyd CE, Mugnier M, Dreesen O, Cross GAM, Papavasiliou FN. Telomere Length Affects the Frequency and Mechanism of Antigenic Variation in Trypanosoma brucei. Hill KL, editor. PLoS Pathog. 2012 Aug 30;8(8):e1002900. doi:10.1371/journal.ppat.1002900
