Atpγs

Radioactive nucleotides were from PerkinElmer Life Sciences (Waltham, Massachusetts). Unlabeled ATP was from Cytiva (Marlborough, MA). ATPγS was from Roche (Basel, Switzerland). DNA-modification enzymes were from New England BioLabs (Ipswich.Massachusetts). DNA oligonucleotides were from Integrated DNA Technologies (Coralville, Iowa). Protein concentrations were determined with the Bio-Rad Labs (Hercules, California) Bradford Protein stain using bovine serum albumin as a standard. Streptavidin-coated Dynabead M-280 magnetic beads were purchased from Thermo-Fisher Scientific (Waltham, Massachusetts). Anti-Digoxigenin, the anti-Dig Fab, was from Millipore-Sigma (St. Louis, MO).

Reaction mixtures (100 μL) contained buffer A [20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM DTT, 0.1 mM EDTA, and 10% glycerol (vol/vol)], 0.005% Triton X-100 (vol/vol), 0.2 mg/mL BSA, 10 mM MgCl₂, 2 mM ATP, and 2 mM ATPγS (Roche), an ATP regenerating system (20 mM creatine phosphate and 6 μg creatine kinase), 0.4 μM GFP or GFP fusion protein, 3.0 μM GroEL_Trap and 1 μM ClpB or Hsp104. GroEL_Trap is a mutant form of GroEL that binds but does not release unfolded proteins and was included in the reactions to prevent the GFP fusion proteins from refolding (Weber-Ban et al., 1999 (link)). Unfolding was initiated with the addition of ATP, ATPγS, and MgCl₂ and the change in fluorescence signal was monitored over time at 25°C using a Tecan Infinite M200Pro plate reader. Excitation and emission wavelengths were 395 and 510 nm, respectively. For K_M and V_max determinations, substrate concentrations were varied between 0.1 and 10 μM while keeping ClpB and Hsp104 concentrations constant at 1 μM. GroEL_Trap was varied between 1 and 5 μM depending on the substrate concentration. Unfolding rates were determined from the initial linear decrease in fluorescence intensities of the GFP fusion proteins. Michaelis-Menten analysis was performed using the non-linear regression analysis in Prism 7.0a for Mac OS X, GraphPad Software, La Jolla California USA (http://www.graphpad.com).

Radioactive nucleotides were from Perkin Elmer and unlabeled nucleotides were from GE Health-care. Protein concentrations were determined using the Bio-Rad Bradford Protein stain using BSA as a standard. Purification of S. cerevisiae Pol α-primase, Pol ε, CMG, the C-terminal half of Ctf4 (residues 471–927), and full length Ctf4 were purified according to previously published procedures (Georgescu et al., 2014 (link); Langston et al., 2014 (link)). The C-terminal half of Ctf4 is necessary and sufficient for Ctf4 binding to CMG and Pol α-primase, as shown previously (Simon et al., 2014 (link)). Oligonucleotides were from IDT (Integrated DNA Technologies). ATPγS used for experiments in this report were purchased from Roche (catalog 11162306001). In experiments testing ATPγS from other companies, ATPγS was purchased from Sigma-Aldrich (catalog A1388) and Tocris Bioscience (catalog 4080).

Purified proteins were incubated at 4 °C for 1 h in buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 2 mM MgCl₂, and 0.1% Triton X-100) supplemented with 10 μM ATPγS (11162306001, Roche). MagStrep type 3 XT beads or 3xFLAG agarose beads were incubated with purified proteins for 1 h at 4 °C. Samples were eluted in BXT buffer or Tris-buffered saline (50 mM Tris-HCl pH 7.5 containing 150 mM NaCl) containing 100 μg/mL 3xFLAG peptide.

To observe the binding distribution of yPCNA on DNA, 0.8 nM yRFC was mixed with 1 nM yPCNA (concentrations reported for the trimer) in BSA buffer supplemented with 100 μM ATPγS (Roche). The yPCNA-yRFC(ATPγS) complex was injected into flowcells containing single-tethered DNA curtains, and incubated for 6 minutes at room temperature. Following this incubation, yPCNA was labeled in situ by injecting 1.5 nM of biotinylated anti-FLAG antibodies conjugated to fluorescent streptavidin-conjugated quantum dots (QDs, emitting at 705 nm, Thermo-Fisher). The antibodies and QDs were pre-conjugated in a test tube on ice prior to injection into the flowcell.
The same protocol was used to measure yPCNA diffusion dynamics with the flowing modifications. First, 1 mM ATP was used instead of 100 µM ATPγS during the yPCNA-yRFC(ATP) incubation. Second, 5 minutes after antibody-coupled QDs were injected into the flowcell, residual yRFC and free QDs were washed out by injecting 200 μL of BSA buffer +300 mM NaCl over 1 minute. yPCNA diffusion data was acquired for 10 minutes after this yRFC washing step.