Direct involvement of the TEN domain at the active site of human telomerase

Abstract

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer Kd, supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5′-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3′-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.

Telomerase Recruitment Requires both TCAB1 and Cajal Bodies Independently

“…After 48 h, the cells were harvested with ice-cold phosphate-buffered saline (PBS). Expression constructs for hTR, hTERT, or hTERT/dyskerin combined were constructed in pApex parental vector (17). G414C and the NAAIRS mutations at amino acid positions 122 to 127 and 176 to 181 and the hTERT R132D mutant were constructed with the Stratagene Quikchange II XL site-directed mutagenesis kit according to the manufacturer's instructions using wild-type (WT) hTR and hTERT, respectively, as the templates; the hTERT mutant with changes at position 170 to 175 (hereinafter called the ϩ170 mutant in line with previous nomenclature [2]) has been previously described (17).…”

“…Expression constructs for hTR, hTERT, or hTERT/dyskerin combined were constructed in pApex parental vector (17). G414C and the NAAIRS mutations at amino acid positions 122 to 127 and 176 to 181 and the hTERT R132D mutant were constructed with the Stratagene Quikchange II XL site-directed mutagenesis kit according to the manufacturer's instructions using wild-type (WT) hTR and hTERT, respectively, as the templates; the hTERT mutant with changes at position 170 to 175 (hereinafter called the ϩ170 mutant in line with previous nomenclature [2]) has been previously described (17). G 1 /S-phase cell cycle synchronization was achieved by incubating cells with 2 mM thymidine (Sigma) for 16 h, releasing for 9.5 h, then incubating with 2 g/ml aphidicolin (Sigma) for 16 h, followed by release for 4 h. Progress through the cell cycle was assessed by DNA content at selected time points by permeabilizing cells on ice for 60 min with 3% (vol/vol) Triton X-100 in the presence of 500 g of RNase and 25 g of propidium iodide in a total volume of 250 l and then analying on a FACSDiva fluorescence-activated cell sorter (Becton Dickinson).…”

“…To test this hypothesis directly, we constructed one of these DAT mutants (changes at position 122 to 127, hereafter called the ϩ122 mutant in line with previous nomenclature [2]) and, using ChIP, tested its ability to localize to telomeres. In conjunction with these experiments, we tested a mutant hTERT with a NAAIRS substitution in a nearby region of the hTERT N terminus at position 170 to 175, which we recently demonstrated to have a severe defect in catalytic activity due to a deficiency in positioning the 3= end of the primer in the active site (17). This mutant (the ϩ170 mutant) was originally reported to fail to immortalize normal human cells upon exogenous expression, and this was thought to arise from the fact that it exhibited no in vitro catalytic activity (2).…”

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

“…5) directly affects primer-template alignment. Mutation of residues 170-175 in the TEN domain of human TERT has recently been reported to alter positioning of the 39 end of the primer in the enzyme active site, perhaps by affecting required conformational changes (Jurczyluk et al, 2011). However, in that case, the catalytic defect is independent of alterations in primer binding.…”

“…Mutations thought to disrupt the S. cerevisiae anchor-site interaction impair catalytic function and processivity on specific primers in vitro (Lue and Li, 2007). The TEN domain of human TERT might also mediate positioning of the 39 end of the primer in the active site in a manner that is independent of the anchor-site interaction defined above (Jurczyluk et al, 2011).…”