Abstract
Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.
Highlights
During every round of eukaryotic cell division, a small amount of DNA is lost from the ends of each chromosome (Olovnikov, 1973; Watson, 1972)
If the resulting ternary complex (TERT:DNA:deoxyribonucletide triphosphate (dNTP)) is in the proper orientation, telomerase reverse transcriptase (TERT) will catalyze the formation of a phosphodiester bond and extend the telomere by one nucleotide (Figure 1A, state C1)
We characterized each step of the TERT catalytic cycle for single nucleotide insertion
Summary
During every round of eukaryotic cell division, a small amount of DNA is lost from the ends of each chromosome (Olovnikov, 1973; Watson, 1972). Telomeres will eventually reach a critically short length, causing cells to undergo apoptosis or become senescent (Hayflick and Moorhead, 1961; Meyerson, 1998) Because telomerase plays such a fundamental role in the temporal regulation of cell division, aberrations in telomerase are implicated in numerous human diseases. We kept our tcTERT kinetics within a single turnover (i.e. insertion) regime, and, wherever possible, complemented the kinetic results with human telomerase studies to characterize the catalytic cycle of telomerase Using this combined approach, we have elucidated the role of conserved telomerase active site residues and determined the mechanisms of fidelity and rNTP discrimination
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