Abstract
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′.
Highlights
Telomeres are DNA/protein complexes located at chromosome ends
Only when undigested chromosomes are separated by Pulsed-Field Gel Electrophoresis (PFGE) can the telomere G-overhang be detected
The telomere G-overhang is clearly observed on minichromosomes in this assay, as T. brucei has ~ 100 minichromosomes that all migrate to approximately the same position in PFGE, the signal is more concentrated
Summary
Telomeres are DNA/protein complexes located at chromosome ends. The specialized telomere structure masks the natural chromosome ends, preventing them from being recognized as DNA breaks by the DNA damage repair machinery; it is essential for maintaining chromosome stability[1]. Our results clearly indicate that telomerase-mediated telomere elongation is a key factor determining the normal telomere G-overhang terminal sequences in T. brucei.
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