Telomere end processing and telomere stability maintenance in trypanosomes

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Project Summary Telomere, the protein/DNA complex at the chromosome end, is essential for keeping eukaryotic cells proliferative. Conventional DNA polymerases cannot fully replicate the linear DNA ends, and most eukaryotes use telomerase to synthesize the telomere G-rich strand de novo. The terminal telomere G-rich 3’ overhang (G3OH) is essential for telomerase-mediated G-strand synthesis and formation of the T-loop structure that is critical for protection of the natural chromosome ends from illegitimate DNA processes. In addition, the length of G3OH needs to be regulated in order to avoid excessive telomeric recombination. Generation of the telomere G3OH involves resection of the telomere 5’ end by exonucleases after the conventional DNA replication, G- strand synthesis by telomerase, and C-strand filled-in. In higher eukaryotes, these telomere end processes are regulated by OB fold-containing telomere ssDNA-binding proteins, which are absent in the kinetoplastid parasite Trypanosoma brucei. Rather, we have found that T. brucei PolIE, a telomere protein and an A family DNA polymerase, suppresses telomerase-mediated G-strand extension, helps ensure proper telomere C-strand synthesis, and suppresses telomeric recombination. T. brucei causes sleeping sickness in humans and regularly switches its major surface antigen, VSG, to achieve immune evasion. VSGs are monoallelically expressed exclusively from loci immediately upstream of the telomere. We have shown that telomere proteins not only are essential for T. brucei cell proliferation but also regulate VSG monoallelic expression and switching. However, other than the fact that telomerase can synthesize the telomere G-strand DNA, mechanisms of telomere end processing and its regulation are poorly understood in T. brucei. In this project, we aim to investigate mechanisms of T. brucei telomere end processing regulation and telomere stability maintenance. In Aim 1, we will investigate how PolIE suppresses the telomerase action and whether it also affects the ending nucleotides of the two telomere strands. The telomere C-strand fill-in in T. brucei presumably requires a DNA polymerase. Since PolIE is a DNA polymerase, in Aim 2.1, we will test whether its DNA polymerase domain is critical for its role in telomere end processing regulation. We have also identified Primase-Polymerase-like protein 2 (PPL2, with a translesion DNA synthesis activity) as a telomere protein and found that PPL2 depletion dramatically elongates the telomere G3OH length. In Aim 2.2, we will investigate whether PPL2 is important for telomere C-strand fill-in and whether this function is PolIE-dependent. T. brucei does not have the non- homologous end joining (NHEJ) machinery, while dysfunctional telomeres are frequently fused through NHEJ in higher eukaryotes. In Aim 3, we will investigate whether homologous recombination and Microhomology- mediated end joining are major pathways for telomeric recombination in WT and PolIE-depleted cells, which will help reveal illegitimate processes that threaten the natural chromosome ends in T. brucei. Our studies will help us better understand the telomere protein evolution and contribute to future development of anti-parasite agents.