Multiple DNA-binding sites in Tetrahymena telomerase

Abstract

Telomerase is a ribonucleoprotein enzyme that maintains chromosome ends through de novo addition of telomeric DNA. The ability of telomerase to interact with its DNA substrate at sites outside its catalytic centre ('anchor sites') is important for its unique ability to undergo repeat addition processivity. We have developed a direct and quantitative equilibrium primer-binding assay to measure DNA-binding affinities of regions of the catalytic protein subunit of recombinant Tetrahymena telomerase (TERT). There are specific telomeric DNA-binding sites in at least four regions of TERT (the TEN, RBD, RT and C-terminal domains). Together, these sites contribute to specific and high-affinity DNA binding, with a K(d) of approximately 8 nM. Both the K(m) and K(d) increased in a stepwise manner as the primer length was reduced; thus recombinant Tetrahymena telomerase, like the endogenous enzyme, contains multiple anchor sites. The N-terminal TEN domain, which has previously been implicated in DNA binding, shows only low affinity binding. However, there appears to be cooperativity between the TEN and RNA-binding domains. Our data suggest that different DNA-binding sites are used by the enzyme during different stages of the addition cycle.

Figure 1.

Rate of dissociation from recombinant Tetrahymena telomerase of DNA primers of different permutations. (A) A bind-and-chase telomerase activity assay (described in the text) was used to measure the dissociation of primer 18GTT (Table 1). The pattern of extension of 18GTT and the chase primer 37TTG alone, when incubated with ddTTP and [α-³²P]dGTP, are shown in the first two lanes. As a control to ensure that the excess of 37TTG did not allow the enzyme to rebind the 18GTT, both primers were preincubated with enzyme simultaneously prior to telomerase extension (‘chase control’). LC: ³²P-labelled 100-mer DNA oligonucleotide used as a recovery and loading control. (B) The amount of signal from products of 18GTT was normalized to the total telomerase product signal and plotted versus time of chase. Two experiments utilizing the same primer (18GTT) are shown. (C) The curves shown in B were fit with a single exponential equation (see ‘Experimental Procedures’) to give the off-rate (k_off) and half-life (t_1/2) of a set of 18 or 20 nt oligonucleotides of different permutations (Table 1). The mean ± SD of two to four experiments is shown.

Figure 2.

A DNA-binding assay to directly measure DNA primer affinity to telomerase. (A) The indicated concentrations of biotinylated telomeric (18GTT) oligonucleotide or 250 nM non-telomeric control (Bio-PBR) were incubated with ³⁵S-labelled full-length recombinant telomerase (TERT + RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: ³³P-labelled PBR48-Bio oligonucleotide, used as a loading and recovery control. (B) Primer extension activity of purified recombinant Tetrahymena telomerase, using biotinylated or non-biotinylated 18GTT primer at the indicated concentrations. ‘n + 1’ is the first nucleotide added to each primer; ‘LC’ is a ³²P-labelled 100-mer DNA oligonucleotide used as a recovery and loading control. (C) Binding or activity were quantified from gels such as those in A and B, and normalized to the highest value. Filled square, binding of Bio-18GTT to telomerase; filled circle, activity using Bio-18GTT; open square, activity using 18GTT. The mean of three to four independent experiments is plotted; error bars = SD.

Figure 3.

DNA interacts with telomerase independently of telomerase RNA. (A) The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with ³⁵S-labelled full-length recombinant TERT (-telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 20% of the starting material in the bound lanes. LC: ³³P-labelled PBR-48-Bio oligonucleotide, used as a loading and recovery control. (B) Quantification of data in A, together with that in Figure 2A, to show increase in K_d in the absence of telomerase RNA. Filled square, binding of Bio-18GTT to TERT + RNA, filled circle, binding of Bio-18GTT to TERT–RNA. The mean of three to four independent experiments is plotted; error bars = SD.

Figure 4.

Summary of binding of oligonucleotide Bio-18GTT to fragments of TERT. At the top is a representation of Tetrahymena thermophila TERT, with conserved protein motifs shown as coloured boxes. The names of the fragments and the amino acids of TERT which they encompass are shown on the left. On the right are the binding constants of DNA and TERT in the presence or absence of telomerase RNA (mean ± SD). *This K_d was measured using crosslinking; n.d. = not determined.

Figure 5.

Crosslinking of 5-iodo-deoxyuridine substituted 20GTT to TERT and fragments of TERT. (A) Sequence of the four oligonucleotides used for crosslinking in B–F; ^IU = 5-iodo-deoxyuridine. (B) ³²P-labelled DNA (15 nM) was crosslinked to full-length TERT in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. Crosslinking was carried out in the presence or absence of telomerase RNA. (C) ³²P-labelled DNA (90 nM) was crosslinked to N519 in the absence of competitor or in the presence of a 55-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (D) ³²P-labelled DNA (15 nM) was crosslinked to C598 in the absence of competitor or in the presence of a 333-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (E) ³²P-labelled DNA (100 nM) was crosslinked to RBD in the absence of competitor or in the presence of a 50-fold excess of cold telomeric (20GTT) or non-telomeric (PBR) competitor primer. (F) ³²P-labelled DNA (50 nM) was crosslinked to full-length TERT, ΔTEN or a 1:1 mixture of both proteins in the presence of a 100-fold excess of cold non-telomeric (PBR) competitor primer. The upper panel shows the signal from ³²P-labelled DNA only (with a piece of X-ray film between the gel and phosphorimager screen to shield ³⁵S signals); the lower panel shows signals from both ³²P and ³⁵S-labelled proteins, to ensure the use of an equimolar mixture of proteins.

Figure 6.

The TEN domain of TERT binds to DNA oligonucleotides with low affinity. (A) DNA immobilization assay using in vitro translated TEN protein. The indicated concentrations of biotinylated 18GTT oligonucleotide were incubated with ³⁵S-labelled TEN protein (+600 nM telomerase RNA) and recovered on NeutrAvidin beads. ‘Input’ represents 40% of the starting material in the bound lanes. LC: ³³P-labelled PBR48-Bio oligonucleotide, used as a loading and recovery control. (B) Crosslinking assay using bacterially expressed, purified TEN protein. The indicated concentrations of TEN protein were crosslinked to 5 nM ³²P-labelled I(1)20GTT DNA.

Figure 7.

Test of RNA-binding ability of TERT fragments. The indicated fragments of TERT were in vitro translated in the presence of ³⁵S-methionine and ³²P-labelled telomerase RNA and then immunoprecipitated with an antibody recognizing a FLAG peptide on TERT. The upper panel shows input material; the lower panel shows the bound fractions.