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 K(d), 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.

Figure 1.

A mutant of the hTERT TEN domain with reduced activity and processivity, and dramatically increased primer K_m. (a) Schematic of the domain structure of hTERT, showing locations of the telomerase essential N-terminal domain (TEN), RNA binding domain (RBD), reverse transcriptase domain (RT) and C-terminal domain (C-term). Coloured boxes indicate conserved sequence motifs. Dots beneath the protein indicate locations of the fifteen NAAIRS substitutions examined in this study. (b) Direct telomerase activity assay of NAAIRS substituted telomerase reconstituted in rabbit reticulocyte lysates, and endogenous telomerase from 293 cells (‘end’). LC: labelled 100 nt oligonucleotide as a recovery and loading control; +4: position of primer (18GGG) with the addition of 4 nt. (c) Quantitation of the relative activity, repeat addition processivity (RAP) and primer K_m (for 18GGG) of the panel of NAAIRS mutants. Values are the mean ± standard deviation of three independent experiments.

Figure 2.

hTERT mutant +170 has a defect in DNA binding affinity. Biotinylated telomeric primers were incubated with wt or mutant telomerase reconstituted in 293T cells. The amount of telomerase recovered on NeutrAvidin beads was quantitated by dot blotting with a ³²P-labelled probe against hTR. The top row is a standard curve of in vitro transcribed hTR. The middle panel represents titrations of the indicated primers, at concentrations of 250, 50, 25, 5, 2.5, 1.25, 0.62, 0.31, 0.15 nM, 78, 39 and 0 pM (wt) or 5, 2.5, 1, 0.5, 0.25 µM, 50, 25, 5, 2.5, 1.25, 0.63 and 0 nM (+170). On the bottom left is binding of telomerase to a non-telomeric DNA control (Bio-PBR), and 20% of the input for each experiment is shown on the bottom right. Note that all panels are from the same exposure of a single dot blot, but are separated for ease of labelling.

Figure 3.

hTERT mutant +170 retains wt global structure. (a) wt and mutant telomerase reconstituted in rabbit reticulocyte lysates were fractionated over parallel 10–40% glycerol gradients, and the fractions subjected to a direct telomerase assay with primer 18GGG. The figure represents two different exposures of a single gel. LC: labelled 100 nt oligonucleotide as a recovery and loading control; +4: primer plus 4 nt. The relative activity in each fraction is plotted on the right. (b) Recombinant hTERT was immunoprecipitated with a FLAG tag antibody, and the recovery of hTR measured by dot blot with a probe against hTR (top panel). Triplicate experiments are shown (A–C). The bottom panel is an SDS–PAGE gel showing the yield of ³⁵S-labelled hTERT protein from each immunoprecipitate of wt or mutant +170 (m) telomerase. (c) WI38 VA13/2RA human fibroblasts were transfected with plasmids encoding hTR and wt or mutant hTERT. Telomerase was immunoprecipitated with an antibody against hTERT. Cell lysate (L) and purified enzyme (E) were electrophoresed on a northern blot for hTR (top panel) or a western blot for hTERT (bottom panel). (d) 293T human embryonic kidney cells were transfected with plasmids encoding hTR and wt or mutant hTERT. Telomerase was immunoprecipitated with an antibody against hTERT and detected on a dot blot for hTR (top panel) or a western blot for hTERT (bottom panel).

Figure 4.

Increased K_m of mutant +170 is not attributable to a defect in translocation rate or interaction with 5′-end of primer. (a) Direct telomerase activity assay with wt or mutant recombinant telomerase using a titration of primer 18GGG, in the presence of ³²P-dTTP and no other nucleotide, in order to measure activity in the absence of translocation. LC: labelled 100 nt oligonucleotide as a recovery and loading control; +1: primer plus 1 nucleotide. (b) Activity assay in the presence or absence of primer 5TTA (5′-GGTTA-3′, 50 µM), with ³²P-dGTP alone (right four lanes) or ³²P-dGTP, dATP and dTTP (left four lanes), using wt or mutant +170 (m) recombinant telomerase. (c) Activity assay using a titration of primer 5GGT and recombinant telomerase. LC: labelled 100 nt oligonucleotide as a recovery and loading control; +3: primer plus 3 nt.

Figure 5.

Proposed kinetic scheme for human telomerase. (a) Alignment of an elongated 18 nt DNA primer (black) with the RNA template (red) before (top) and after (bottom) translocation and addition of two Gs. Numbers 1–6 are assigned to each position of nucleotide addition within a single telomeric repeat. (b) Proposed kinetic scheme for telomerase. E = telomerase, S = DNA substrate, with 3′-permutation of each DNA primer in parenthesis. ES = telomerase–DNA complex. It has been demonstrated that the dissociation rate (k_off) of a particular DNA sequence is the same whether that DNA is the substrate or product of extension (47), so only one k_off has been included for each sequence. Note that the rate constant for translocation (k_trans) is potentially composed of at least two other rate constants, for RNA–DNA dissociation and realignment, respectively (50). Protein conformational changes and nucleotide association constants are not shown in this scheme. The dashed box indicates the portion of this scheme measured in a reaction with 18GGG as the DNA primer and dTTP as the only nucleotide.

Figure 6.

hTERT mutant +170 is defective in nucleotide polymerization, particularly opposite position 51 of the template. (a) Schematic of alignment of permuted 18 nt DNA primers with the RNA template. Nucleotide position numbers of the template within hTR are shown. Primer DNA sequence is in black uppercase; nucleotides added in reactions with a single radiolabelled nucleotide are indicated in coloured bold lower case. In primer 18ACT, the region of mismatch with the RNA template is underlined. (b) Activity assay measuring polymerization rate at each position of the RNA template, using recombinant telomerase (top panel) or telomerase overexpressed in 293T cells (bottom panel). Assays included the DNA primers indicated, and a single radiolabelled nucleotide: ³²P-dTTP (with 18GGG and 18GGT), ³²P-dATP (with 18GTT) or ³²P-dGTP (with 18TTA, 18TAG and 18AGG). Positions of templated additions of nucleotides are indicated with coloured asterisks. M = marker, ³²P end labelled 18GGG primer. (c) Extension of primer 18ACT by wild type (wt) and mutant +170 (m) recombinant telomerase, in processive (lanes 3 and 4) or non-processive (lanes 5 and 6) reactions. LC: labelled 100 nt oligonucleotide as a recovery and loading control; +1: primer plus 1 nt; +4: primer plus 4 nt. (d) Titration of dGTP in a non-processive reaction with primer 18TTA. The specific activity of the ³²P-dGTP was kept constant over all dGTP concentrations. +1: primer plus 1 nt.

Figure 7.

Models of the location, structure and function of hTERT region 170–175. (a) Proposed function of hTERT amino acids 170–175 in positioning 3′-end of primer in active site. According to this model, this region of the TEN domain (yellow circle) is positioned close to the enzyme active site (white star). As each nucleotide is added to a DNA primer ending in—TTA, the DNA–RNA duplex becomes repositioned so the new 3′-end is in the active site. Depicted in red is the hTERT amino acid homologous to Tetrahymena W187; the latter has been shown to interact with a DNA nucleotide close to the template boundary (28). See text for more detail. (b) Top: crystal structure of the TEN domain from Tetrahymena TERT (19). The amino acids homologous to hTERT region 170–175 are shown in yellow [from sequence alignment in ref. (56)]. Amino acids involved in crosslinking to a DNA primer (Q168, F178, W187) are red (19,28). An amino acid involved in repeat addition processivity (L14) is blue (31). Bottom: homology model of the TEN domain of human TERT. The amino acids mutated in this study (170–175) are in yellow. Amino acids homologous to those shown in Tetrahymena to crosslink to a primer are shown in red (Q169, P188). hTERT region 122–136 is coloured purple [part of the ‘DAT’ domain (32)]. Amino acids involved in repeat addition processivity (L13 and L14) are blue (31). N95, shown to be involved in DNA binding, is green (29). Figures were constructed using PYMOL (57). Note that the colours used in this figure bear no relationship to those used in Figure 1.