Calmodulin affinity resin

Tandem affinity purification of ISW1a (TAP-Ioc3) and Fun30 (TAP-Fun30) was performed as follows: cultures were grown in YPD media, harvested cells were washed once with water. The cells were lysed in buffer E (20 mM Na·HEPES pH 7.5, 350 mM NaCl, 10% glycerol, 0.1% Tween, and 0.5 mM DTT) and protease inhibitors by grinding in the presence of liquid nitrogen. Lysates were clarified at 40,000 × g at 4 °C for 1 h. Cleared lysates were incubated with IgG-Sepharose (GE Healthcare) at 4 °C for 2 h and eluted by TEV protease (Invitrogen) cleavage at 4 °C overnight. The elutions were incubated with calmodulin affinity resin (Agilent Technology) in buffer E plus 2 mM CaCl₂ at 4 °C for 2 h and eluted in buffer E plus 10 mM EGTA.
ISW2 (2xFLAG-Isw2, YTT480^{32 (link)}) was purified as follows: cleared lysate was incubated with Anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4 °C for 1 h and eluted with 0.1 mg/ml 3X FLAG peptide (Sigma-Aldrich). E-buffer (20 mM Na·HEPES pH 7.5, 350 mM NaCl, 10% glycerol, 0.1% Tween, and 0.5 mM DTT) was used during the entire purification.
Purified proteins were concentrated with VIVASPIN concentrators (Sartorius) and dialyzed against E-Buffer with 1 mM DTT. Subunit compositions were confirmed by SDS-PAGE (Supplementary Fig. 1a) and mass spectrometry. For full scan images of all gels presented in this study see Source Data.

Cell powder from 20 l of yeast culture was resuspended in buffer 1 (25 mM HEPES-KOH pH 7.6, 0.05% NP-40, 10% glycerol, 2 mM beta-mercaptoethanol) supplemented with 0.1 M potassium chloride and protease inhibitors. Potassium chloride concentration was increased to 0.5 M before subjecting the lysate to ultracentrifugation at 235,418g (45,000 r.p.m.) for 60 min at 4 °C. Cleared lysates were supplemented with 2 mM calcium chloride and incubated for 2 h at 4 °C with 5 ml of Calmodulin Affinity Resin (Agilent) pre-equilibrated in buffer 1 plus 0.4 M potassium chloride and 2 mM calcium chloride. Beads were subsequently washed with 80 ml of buffer 1 supplemented with 0.4 M potassium chloride and 2 mM calcium chloride, and bound proteins were eluted using buffer 1 supplemented with 0.4 M potassium chloride, 2 mM EDTA and 2 mM EGTA. Pooled fractions were concentrated using a 100,000-molecular weight cutoff (MWCO) spin column and subsequently separated on a Superdex 200 16/600 gel filtration column (GE Healthcare) in buffer 1 supplemented with 0.15 M potassium chloride. Peak fractions were pooled and concentrated.

In the pulldown assays with the replisome factors, 0.5 μM of GST-Pob3Spt16 was incubated with 1 μM of Pol α, Ctf4, GINS, Cdc45 and Tof1-Csm3, on ice for 30 min in a total volume of 50 μl. Proteins were immobilized on 15 μl of Protino™ Glutathione Agarose 4B (Macherey-Nagel) for 90 min at 4°C. Similarly, CBP-Csm3Tof1, GST fusion proteins (Tof1_N, Tof1_M, Tof1_C) and GST (1.5 μM) were incubated with prey proteins (fl FACT and FACT truncations, 3 μM) and immobilized on 15 μl of Calmodulin Affinity Resin (Agilent) or Protino™ Glutathione Agarose 4B (Macherey-Nagel) for 90 min at 4°C. Following, beads were washed 3× with buffer G (50 mM HEPES-NaOH, pH 7.6, 300 mM NaCl, 10% (v/v) glycerol, 0.05% NP-40, 1 mM DTT, 1 mM EDTA) for GST pulldowns or buffer C (25 mM HEPES-NaOH, pH 7.6, 300 mM NaCl, 10% (v/v) glycerol, 0.05% NP-40, 1 mM DTT, 2 mM CaCl₂) for CBP pulldowns. Proteins were incubated in the buffer C + 5 mM EDTA, 5 mM EGTA or buffer G + 20 mM reduced glutathione, for 10 min on shaker at 4°C, 1000 rpm, eluted by centrifugation (500 × g, 4°C, 1 min) and analyzed by SDS-PAGE.