Leishmania mexicana telomeres at high resolution: Ku80, TERT, and alternative lengthening mechanisms

Telomeres are known to be important for Leishmania biology but the mechanistic of how the process of telomere maintenance contributes to genome stability remains an unanswered question. Their maintenance is most commonly facilitated by the telomerase ribonucleoprotein complex that elongates telomeres countering their natural shortening due to the incomplete DNA replication in each cell cycle. In some organisms, telomere maintenance is achieved through telomerase-independent mechanisms, such as the Alternative Lengthening of Telomeres (ALT) pathways described in yeasts with dysfunctional telomerase and some rare telomerase-negative human cancer cells. Molecular markers for the ALT pathway include presence of the heterogeneous (in their length and sequence) telomeres, high level of telomeric exchange between the sister chromatids, increased expression of Rad51 and associated proteins, and occurrence of extrachromosomal telomeric repeats that can be present in either linear or circular form.

Here, we used third-generation sequencing techniques in combination with other approaches and analyzed telomeres of L. mexicana at unprecedented high-level resolution. We demonstrate that Ku80 ablation-driven telomere elongation varies between chromosomes, possibly due to the chromosome-specific recombination rates, which are sequence/content dependent and associated with the structure of the telomeric tandemly repeated sequence, TTAGGG. Moreover, this telomere length heterogeneity is accompanied by an increased level of C-circles, a subclass of circular telomeric DNA highly specific for ALT activity.

Our findings underscore that L. mexicana promastigotes have an inherent ability to utilize ALT, and the loss of Ku80 and/or TERT further enhanced this trait. These proteins work together to maintain telomere integrity, inhibit recombination, and stabilize telomere lengths. Our data suggest that ALT may be a fundamental and readily activated feature of Leishmania biology, and that telomere regulation in this organism significantly differs from what has been observed in other eukaryotic model species, including iconic T. brucei.