DNA pulldowns

Biotinylated telomeric and control DNA for the DNA pulldown for detection of telomeric interactors was prepared as previously published^16,39,40. In short, 25 µl of 10-mer repeat oligonucleotides of either telomeric or control sequence were mixed 1:1 with 25 µl of their respective reverse complement oligonucleotide and 10 µl annealing buffer (200 mM Tris-HCl, pH 8.0, 100 mM MgCl2, 1 M KCl). The mixture was brought to 100 µl final volume with H₂O, heated at 80 °C for 5 min, and left to cool. Once at room temperature (RT), the samples were supplemented with 55 µl H₂O, 20 µl 10x T4 DNA ligase buffer (Thermo Scientific), 10 µl PEG 6000, 10 µl 100 mM ATP, 2 µl 1 M DTT and 5 µl T4 Polynucleotide Kinase (NEB, 10 U/µl, #M0201) and left at 37 °C for 2 h to concatenate. Finally, 4 µl of T4 DNA Ligase (Thermo Scientific, 5 WU/µl, #EL0011) were added and the samples incubated at RT overnight for ligation and polymerization. The ligation process was monitored by running 1 µl of the reaction on a 1% agarose gel. The samples were cleaned by phenol-chloroform extraction. For this, 1 vol. of H₂O and 200 µl of Phenol/Chloroform/Isoamyl Alcohol (25:24:1; pH 8; Invitrogen, # 15593049) was added to the mixture, vortexed and centrifuged at 16,000 x g for 2 min. After centrifugation the aqueous phase was transferred to a fresh tube and the DNA precipitated by addition of 1 ml 100% Ethanol and incubation at −20 °C for 30 min. Afterwards the suspension was centrifuged at 16,000 x g for 45 min at 4 °C. The resulting DNA pellet was resuspended in 74 µl H₂O and 10 µl 10x Klenow-fragment reaction buffer (Thermo Scientific), 10 µl 0.4 mM Biotin-7-dATP (Jena Bioscience, #NU-835-BIO) and 6 µl Klenow-Fragment exo- polymerase (Thermo Scientific, 5 U/µl, # EP0422) added. Biotinylation was carried out by incubation at 37 °C over night. The reaction was cleaned up by size-exclusion chromatography using MicroSpin Sephadex G-50 columns (GE Healthcare, #GE27-5330-01).

Biotinylated DNA and Dynabeads™ MyOne™ Streptavidin C1 (Thermo Scientific, #65001) were mixed with PBB buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5% NP 40, 5 mM MgCl₂, 1 mM DTT) and incubated at room temperature for 15 min on a rotating wheel to immobilize the DNA on the beads. After three washes with PBB buffer, the DNA coupled beads were resuspended in PBB buffer and Salmon sperm (10 mg/ml, Ambion, #AM9680) was added 1:1000 as competitor for unspecific DNA binding. The pulldowns were performed with different amounts of protein extract (see below) and incubated at 4 °C on a rotating wheel for 90 min. Following incubation the beads were washed three times with PBB buffer and resuspended in 1x Loading buffer (4x NuPAGE LDS sample buffer, Thermo Scientific, #NP0008) supplemented with 100 mM DTT. For elution, the samples were boiled at 70 °C for 10 min and afterwards loaded on a gel and processed as indicated above for MS, or below for western blot. In pulldown-MS experiments, the pulldowns were prepared in either technical quadruplicates (LFQ), or technical duplicates (DML) per condition, whereas for western blot all conditions were prepared with one replicate and an input. In all, 200–400 µg of nuclear worm extract and of Escherichia coli extract were used for the mass spectrometry screen and pulldowns of Fig. 1c, respectively. In all, 0.4–0.7 mg of total protein extract were used for the pulldowns shown in Fig. 1d–f. Four-hundred micrograms of E. coli extract was used in DNA-binding domain pulldowns in Fig. 6e.