Abstract
Normal human stem cells rely on low levels of active telomerase to sustain their high replicative requirements. Deficiency in telomere maintenance mechanisms leads to the development of premature aging diseases, such as dyskeratosis congenita and aplastic anemia. Mutations in the unique "insertion in fingers domain" (IFD) in the human telomerase reverse transcriptase catalytic subunit (hTERT) have previously been identified and shown to be associated with dyskeratosis congenita and aplastic anemia. However, little is known about the molecular mechanisms impacted by these IFD mutations. We performed comparative functional analyses of disease-associated IFD variants at the molecular and cellular levels. We report that hTERT-P721R- and hTERT-R811C-expressing cells exhibited growth defects likely due to impaired TPP1-mediated recruitment of these variant enzymes to telomeres. We showed that activity and processivity of hTERT-T726M failed to be stimulated by TPP1-POT1 overexpression and that dGTP usage by this variant was less efficient compared with the wild-type enzyme. hTERT-P785L-expressing cells did not show growth defects, and this variant likely confers cell survival through increased DNA synthesis and robust activity stimulation by TPP1-POT1. Altogether, our data suggest that multiple mechanisms contribute to cell growth defects conferred by the IFD variants.
Highlights
In the present study, using HEK 293 and HeLa cells overexpressing the telomerase variants, we found that human telomerase reverse transcriptase catalytic subunit (hTERT)-P721R and hTERT-P785L displayed altered levels of telomerase activity, and under limiting amounts of dGTP all in fingers domain” (IFD) variants showed lower levels of repeat addition processivity (RAP) compared with the with the control sample (WT) enzyme
Data from patient samples, rabbit reticulocyte lysates (RRLs), and immunopurified telomerase from cell extracts indicated that P721R, T726M, and R811C hTERT variants have slight defects in enzyme activity and processivity as assessed by telomeric repeat amplification protocol (TRAP) or DPE experiments (26 –30, 32)
Based on our DPE data showing that hTERT-P785L is the only mutant enzyme for which telomerase activity is stimulated by TPP1-POT1 overexpression, we investigated whether recruitment of the IFD variants to the telomeres can be enhanced by overexpressing TPP1-POT1 (Fig. 3C)
Summary
To ensure that the altered levels of observed telomerase activity and RAP were not due to defects in holoenzyme assembly, we performed a co-IP experiment using 3XFLAG-tagged hTERT-WT and hTERT variants followed by the detection of bound hTR using quantitative RT-PCR (Fig. 1, F and G). Based on our DPE data showing that hTERT-P785L is the only mutant enzyme for which telomerase activity is stimulated by TPP1-POT1 overexpression, we investigated whether recruitment of the IFD variants to the telomeres can be enhanced by overexpressing TPP1-POT1 (Fig. 3C).
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