Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei

Trypanosoma brucei causes fatal human African trypanosomiasis and evades the host immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. Telomere length and telomere proteins play important roles in regulating VSG silencing and switching. T. brucei telomerase plays a key role in maintaining telomere length, and T. brucei telomeres terminate in a single-stranded 3' G-rich overhang. Understanding the detailed structure of the telomere G-overhang and its maintenance will contribute greatly to better understanding telomere maintenance mechanisms. Using an optimized adaptor ligation assay, we found that most T. brucei telomere G-overhangs end in 5' TTAGGG 3', while a small portion of G-overhangs end in 5' TAGGGT 3'. Additionally, the protein and the RNA components of the telomerase (TbTERT and TbTR) and TbKu are required for telomere G-overhangs that end in 5' TTAGGG 3' but do not significantly affect the 5' TAGGGT 3'-ending overhangs, indicating that telomerase-mediated telomere synthesis is important for the telomere G-overhang structure. Furthermore, using telomere oligo ligation-mediated PCR, we showed for the first time that the T. brucei telomere 5' end sequence - an important feature of the telomere terminal structure - is not random but preferentially 5' CCTAAC 3'.