α 32p dgtp

The human telomerase was overexpressed and purified as previously described [35 (link)]. Purified recombinant shelterin protein complexes (POT1-TPP1-TIN2(1–354), TRF2-RAP1 or POT1-TPP1-TIN2(1–354)-TRF2-RAP1) at concentrations from 100 to 5 nM (20^1/7-fold serial dilution) were incubated with 10 nM telo666 telomeric ligand (S1B Fig) in a total volume of 10 μl telomerase assay buffer [50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl₂, 4 mM 2-mercaptoethanol, 500 μM each of dATP and dTTP, 20 μM dGTP and 20 nM [α-³²P]dGTP (6000 Ci/mmol, PerkinElmer)]. The binding reactions were pre-equilibrated at 30°C for 30 min. Telomeric DNA extension reactions were initiated by the addition of purified recombinant telomerase and incubated at 30° C for an extra 30 min. Reactions were terminated by addition of an equal volume of deionised formamide. After addition of proteinase K (0.5 mg/ml), the reaction mixtures were further incubated at 37° C for 20 min. The reaction products were heat-denatured for 10 min at 95°C and one-half of the reaction volume was resolved (17 W, 65 min) on a pre-electrophoresed (17 W, 60 min) denaturing (7 M urea, 0.5× TBE) 8% polyacrylamide (19:1 mono:bis ratio) gel. Gels were subsequently dried onto a positively charged Hybond N+ nylon membrane (GE Healthcare) and phosphorimaged (Typhoon FLA 7000).

DNA oligonucleotides and chemically modified triphosphates. dA^imTP and dU^gaTP were synthesized according to literature precedent,71 (link),82 and dC^aaTP was obtained from Trilink. Oligonucleotides were purchased from Integrated DNA Technologies (IDT), and purified by denaturing (7 M urea) 10 or 20% polyacrylamide gel electrophoresis (PAGE) before use. Details regarding the oligonucleotide sequences and the preparations are provided in the ESI.† α-³²P-dGTP and γ-³²P-ATP were purchased from Perkin Elmer.

Plasmids pPK14640 and pPK14641 were isolated using a QIAfilter maxi kit (Qiagen), and 30 ng was digested using BamHI and HindIII enzymes (NEB) to expose the 3′ ends of the fragment for radiolabeling with [α-³²P]dGTP (PerkinElmer) by Sequenase (Thermo Fisher). The relevant fragments were separated and isolated from a 5% acrylamide-TBE (Tris-borate-EDTA) gel using a QIAquick gel extraction kit (Qiagen). Promoter fragments were incubated with RisR (1 μM) for 25 min in 25 mM potassium phosphate buffer, 30 mM KCl, 5 mM potassium glutamate, 100 μg/mL BSA, and 1 mM DTT at 37°C under anaerobic conditions. DNase I (Worthington) 2 μg/mL in 65 mM MgCl₂ was added for 30 s, and the reaction was terminated with 300 mM acetate and 20 mM EDTA, ethanol precipitated and resuspended in 4 μL urea loading dye, heated to 90°C for 1 min before loading a 7-M urea −8.0% polyacrylamide gel in 0.5× TBE buffer. The G+A ladder for each radiolabeled promoter fragment was achieved by DNA modification using formic acid followed by piperidine cleavage (79 (link)). The gel was visualized in an Amersham Typhoon 5-gel imaging scanner (Cytiva). For some experiments, 5 μM RisR was treated with 32 units of enterokinase (NEB) to remove the N-terminal tag by 2 h of incubation at room temperature under anaerobic conditions in 50 mM potassium phosphate buffer, 100 mM NaCl, 2 mM CaCl₂, and 10% glycerol pH 7.2.

The telomerase assay was as described^{12 (link)}. Reactions (20 μl) contained 1× human telomerase buffer, 1 μM oligonucleotide substrate, and dNTP mix as indicated in the figure legends. Reactions with cellular dNTP concentrations contained 24 μM dATP, 29 μM dCTP, 37 μM dTTP, 5.2 μM dGTP, and 0.3 μM 3000 Ci per mmol [α-³²P] dGTP or [α³²P] dTTP (PerkinElmer) as indicated. Reactions containing the modified dNTPs (Trilink Biotechnologies) substituted for their natural dNTP analog are indicated in the figure legends. The reactions were started by the addition of 3 μl (~35 fmol) of immunopurified telomerase eluent, incubated at 37 °C for 1 h, then terminated with 2 μl of 0.5 mM EDTA and heat inactivated at 65 °C for 20 min. ³²P-end labeled 18-mer loading control (8 fmol) was added to the terminated reactions before purification with an Illustra Microspin G-25 column (GE Healthcare). An equal volume of loading buffer (94% formamide, 0.1× TBE, 0.1% bromophenol blue, 0.1% xylene cyanol) was added to the reaction eluent from the G-25 spin column. The samples were heat denatured for 10 min at 100 °C and loaded onto a 14% denaturing polyacrylamide gel (7 M urea, 1× TBE) and electrophoresed for 90 min at constant 38 W. Samples were imaged using a Typhoon phosphorimager (GE Healthcare).

Telomerase activity was assayed in a total volume of 20 μl at 18° for 30 min, with 10 μl of the telomerase extract in a 1x telomerase reaction buffer (0.05 M Tris-HCl pH 8.0, 1mM spermidine, 1mM DTT), 50 μM dTTP, 50 μM dCTP, 200 mM K-Glu, 0.083 μM [α³²P]-dGTP (PerkinElmer), 0.54 μM dGTP, and a gel purified oligonucleotide, (GGGTGTCT)₂, with a final concentration 1.65 μM. The primer extension reactions were stopped with 200 μl 10 mM Tris-HCl (pH 7.5) and 21 mM EDTA. An 11-mer telomeric primer, which was labeled with [γ³²P]-ATP by T4 Polynucleotide kinase, was added into the stop solution as loading control. Extraction was made with an equal volume of phenol/chloroform:isoamyl alcohol (24:1). An equal volume of 5 M ammonium acetate with 0.1 mg/ml glycogen was added before DNA precipitation with 1 ml 95% ethanol in room temperature for 60 min. The samples were washed with 70% ethanol and dried. Pellets were resuspended in 4 μl Xylene-cyanol dye mix, incubated at 90° for 2 min and loaded on a 10% polyacrylamide/7 M Urea sequencing gel. The gel was run at 1800 V for 1h 40 min in 0.6 x TBE buffer (26.7 mM Tris-Borate and 0.6 mM EDTA), dried and analyzed for radioactive signal with a BioRad Molecular Imager FX PhosphorImager.

Unlabeled nucleotides and dideoxynucleotides were purchased from General Electric Healthcare. [α-³²P]dATP (3000 Ci/mmol), [α-³²P]dGTP and [α-³²P]dCTP (3000 Ci/mmol) were supplied by PerkinElmer Inc. Oligonucleotides were obtained from Sigma-Aldrich and PAGE purified. TP-containing Φ29 DNA (Φ29 TP-DNA) was obtained as described (36 (link)). The oligonucleotide hybridizations to obtain the different dsDNA origins were performed in TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA)) at 8 μM each oligonucleotide in a Biorad (Hercules, CA, USA) MyCycler thermal cycler using the following program, 95°C for 5 min, 93°C for 30 s, 85°C for 10 min, 80°C for 10 min, 75°C for 5 min, 70°C for 5 min, 65°C for 5 min, 45°C for 60 min and then holding at 4°C.