The Telomere Terminal Structure in Trypanosoma brucei

Telomeres are specialized nucleoprotein complexes at the end of linear chromosomes. They are essential for chromosome stability and genome integrity. In most eukaryotes, telomere DNA consists of simple repetitive TG-rich sequences, and there is a single-stranded 3’ G-rich overhang at the very end of the telomere. This telomere G-overhang structure is essential for telomere maintenance: in the presence of telomerase, G-overhang serves as a substrate; in the absence of telomerase, G-overhang can mediate efficient break-induced-repair (BIR) of chromosome ends, an alternative mechanism for telomere maintenance and an important pathway for telomere recombination. Telomere recombination is particularly important for Trypanosoma brucei, a protozoan parasite and the causative agent of Human African Trypanosomiasis. T. brucei evades the host’s immune attack by regularly changing its variant surface glycoprotein (VSG), and homologous recombination is one of several important mechanisms for VSG switching. Therefore, abnormal telomere recombination may affect VSG switching efficiency. So far, the telomere terminal structure in T. brucei is poorly understood, and the only available native in-gel hybridization analysis is not sensitive enough to reveal the details of this structure. To better characterize the T. brucei telomere G-overhang structure, we adopted a ligation-mediated primer extension assay. This assay allowed us to determine both the length and the terminal nucleotide of the telomere G-overhang, and we observed that the majority of T. brucei telomeres have very short G-overhangs that end in 5’ TTAGGG 3’. Such TTAGGG-ending G-overhangs are greatly diminished in cells lacking TERT, the telomerase protein component, and in cells depleted of tbTRF, the duplex telomere DNA binding factor.