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

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′.

Trypanosoma bruceiPolIE suppresses telomere recombination

“…Therefore, we tested whether depletion of PolIE affected the telomere G-overhang structure in T. brucei. WT T. brucei cells have short telomere G-overhangs that are estimated to be 12-nt long or shorter (10,11). Indeed, we only detected a very faint telomere Goverhang signal in WT cells using the native in-gel hybridization analysis (Fig.…”

“…Telomerase uses a G-rich single-stranded 3' DNA end as its substrate (7). Hence, the telomere single-stranded 3' G-rich overhang structure is important for telomerase-mediated telomere synthesis (8)(9)(10)(11). The telomere G-overhang is also critical for the formation of the telomere T-loop structure (12,13), which in turn helps protect the telomere from illegitimate nucleolytic degradation and DNA damage repair processes (2).…”

“…T. brucei telomeres also form a T-loop structure (13). In addition, T. brucei telomere has a short 3' single-stranded G-rich overhang (10,11). The telomere structure and telomere proteins are not only essential for T. brucei genome stability and cell proliferation (36) but are also essential for monoallelic VSG expression (37)(38)(39)(40) and influence the VSG switching frequency (41)(42)(43)(44).…”

“…However, detailed T. brucei telomere synthesis mechanisms are still not clear. We previously found that T. brucei has very short telomere G-overhangs (~12 nts) (10) and the telomerase activity is essential for the telomere G-overhang structure (11), suggesting that resection of the telomere 5' end by an exonuclease, which has not been identified in T. brucei yet, is minimum and/or the telomere C-strand fill-in step is efficient. So far, no protein has been identified to bind specifically to the single-stranded telomere DNA in T. brucei, and how telomere C-strand fill-in is achieved is unknown.…”

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes

“…brucei telomere 3ʹ overhang ends have the sequence 5ʹ TTAGGG 3ʹ, whereas a small part of the overhang has the sequence 5ʹ TAGGGT 3ʹ. Telomerase activity is essential for the maintenance of 5ʹ TTAGGG 3ʹ ends but apparently does not affect the 5ʹ TAGGGT 3ʹ ends [ 50 ]. This finding suggests that the 5ʹ TAGGGT 3ʹ ends can be maintained and/or generated by a potentially telomerase-independent mechanism, such as telomerase-independent telomere lengthening, which involves telomeric recombination [ 51 – 53 ].…”