Gel filtration chromatography of telomeric complexes. (a) Characterization of a reconstituted telomeric complex. Size-exclusion chromatography reveals a sharp peak at 1.3 mL representing a complex of 132-mer DNA coated with 11 POT1-TPP1 heterodimers. The smaller peak (1.8 mL) represents excess POT1-TPP1 heterodimer. (b) Chromatogram of 132-mer telomeric DNA alone. Telomeric complexes were assembled by combining telomeric ssDNA with purified, recombinant POT1-N, POT1, or POT1-TPP1 heterodimers. Thirty µL reactions, containing 160 nM DNA and protein at a 2 × molar excess to POT1 binding sites on the ssDNA substrate, were incubated for fifteen min on ice, then separated on a Superose 6 PC 3.2/30 chromatography column using a SMART system (Pharmacia). Approximately 20 µL were injected on the SMART system and 25 µL fractions were collected.

Negative-stain electron microscopy of telomeric complexes. (a) A typical micrograph of telomeric complexes assembled from 144-nt telomeric DNA saturated with twelve POT1-TPP1 heterodimers. The particles are globular in shape and monodisperse on the grid. Scale bar is 100 nm. (b) 2D class averages of the 144nt-POT1-TPP1 telomeric complexes. The purified complexes were brought to a final concentration of approximately 50 µg/mL in 25 mM Hepes pH 8.0, 150 mM NaCl. Grids were prepared using the sandwich method ⁴³ and stained in 0.75% uranyl formate as described.³⁵ Micrographs were recorded on a 4K × 4K Gatan CCD camera with a Tecnai F20 electron microscope operating at an accelerating voltage of 200 keV and a magnification of 39,000 X. Pixel sizes, after binning, were 5.52 Å per pixel on the object scale. All image processing was done with SPIDER software.⁴⁴ A total of 3664 particles were picked and classified into 57 class averages using reference-free alignment and K-means classification procedures. Class averages are windowed in boxes with 440Å × 440Å dimensions.

Electrophoretic mobility shift assays depicting the ability of POT1-N to coat long tracts of telomeric DNA. ssDNA oligos were synthesized by Integrated DNA Technologies. Oligos ranged from 24-nt to 48-nt and contain four to eight telomere hexameric repeats. Every two hexameric repeats (5’-GGTTAG GGTTAG-3’) is suitable for binding individual POT1 molecules. DNA was 5’-radiolabeled and gel shifts were performed essentially as previously described.³³ For each reaction, a 10 µL solution was prepared containing 400 nM ssDNA spiked with approximately 4% 5’-³²P-labeled DNA. POT1-N was added to each reaction at molar ratios indicated on top of the gel. The molar ratio indicated is calculated for the ratio of protein added per dodecameric repeat (5’-GGTTAG GGTTAG-3’) in the oligonucleotide in each lane. Reactions were conducted in buffer containing 25 mM Tris, pH8.0, 150 mM NaCl, 10 mM DTT and 5% glycerol. After thirty minutes on ice, reactions were analyzed by gel electrophoresis on 6% acrylamide gels in 0.5X TBE buffer. Gels were dried and imaged on a Typhoon phosphorimager (GE Lifesciences).

Electrophoretic mobility shift assays of POT1-N (a), POT1 (b), and POT1-TPP1 (c,d) binding to a 72-mer telomeric DNA substrate. Gel shifts were performed essentially as described in Figure 1. Briefly, 200 nM of a DNA solution (containing ~4% 5’-³²P-labeled DNA) was incubated with increasing concentrations of protein for fifteen min on ice in 10 µL buffer containing 25 mM Hepes, pH 8.0, 150 mM NaCl, 5 mM DTT and 5% glycerol. The protein concentration in each reaction ranged from 0 – 6 µM in 1.2 µM increments. POT1 and POT1-N were expressed as GST-fusion proteins in baculovirus infected insect cells as previously described.³³ TPP1(89–334) was expressed with an N-terminal 6XHis tag. POT1-TPP1 heterodimers were purified after co-expression in insect cells using both affinity tags and size-exclusion chromatography. After incubation, the protein-DNA complexes were subject to electrophoresis in a non-denaturing polyacrylamide gel and dried. In panels a–d, line markers on left side of gel indicate 1,2,3,4,5, or 6 proteins bound to the DNA; arrow indicates position of wells. Panel (e), The relative abundance of each complex was quantified using a Typhoon Imager (GE Lifesciences) and ImageQuant TL Software (GE Lifesciences). For quantification, the number of counts representing ssDNA forming the specific complex with six proteins was divided by the total number of counts in each lane (including the aggregates) and plotted as a percentage.