SYTOX Orange dye

To construct the rolling circle template (Tanner et al., 2009 (link)), the 66-mer 5'-biotin-T₃₆AATTCGTAATCATGGTCATAGCTGTTTCCT-3' (Integrated DNA Technologies, Coralville, IA) was annealed to M13mp18 ssDNA (New England Biolabs) in TBS buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 50 mM NaCl) at 65°C. The primed M13 was then extended by adding 64 nM T7 gp5 polymerase (New England Biolabs) in 40 mM Tris-HCl pH 7.6, 50 mM potassium glutamate, 10 mM MgCl₂, 100 μg/ml BSA, 5 mM dithiothreitol and 600 μM dCTP, dGTP, dATP and dTTP at 37°C for 60 min. The reaction was quenched with 100 mM EDTA and the DNA was purified using a PCR purification kit (Qiagen, the Netherlands). Microfluidic flow cells were prepared as described (Geertsema et al., 2015 (link)). Briefly, a PDMS flow chamber was placed on top of a PEG-biotin-functionalized microscope coverslip (Figure 1—figure supplement 1 inset). To help prevent non-specific interactions of proteins and DNA with the surface, the chamber was blocked with buffer containing 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 50 mM NaCl, 0.2 mg/ml BSA, and 0.005% Tween-20. The chamber was placed on an inverted microscope (Nikon Eclipse Ti-E) with a CFI Apo TIRF 100x oil-immersion TIRF objective (NA 1.49, Nikon, Japan) and connected to a syringe pump (Adelab Scientific, Australia) for flow of buffer.
Conditions for coupled DNA replication under continuous presence of all proteins were adapted from previously described methods (Tanner et al., 2008 (link), 2009 (link)). All in vitro single-molecule experiments were performed at least four times. Briefly, 30 nM DnaB₆(DnaC)₆ was incubated with 1.5 nM biotinylated ds M13 template in replication buffer (25 mM Tris-HCl pH 7.9, 50 mM potassium glutamate, 10 mM Mg(OAc)₂, 40 μg/ml BSA, 0.1 mM EDTA and 5 mM dithiothreitol) with 1 mM ATP at 37°C for 30 s. This mixture was loaded into the flow cell at 100 μl/min for 40 s and then at 10 μl/min. An imaging buffer was made with 1 mM UV-aged Trolox, 0.8% (w/v) glucose, 0.12 mg/ml glucose oxidase, and 0.012 mg/ml catalase (to increase the lifetime of the fluorophores and reduce blinking), 1 mM ATP, 250 μM CTP, GTP and UTP, and 50 μM dCTP, dGTP, dATP and dTTP in replication buffer. Pol III* was assembled in situ by incubating τ₃δδ’χψ (410 nM) and SNAP-labeled Pol III cores (1.2 μM) in imaging buffer at 37°C for 90 s. Replication was initiated by flowing in the imaging buffer containing 6.7 nM Pol III* (unless specified otherwise), 30 nM β₂, 300 nM DnaG, 250 nM SSB₄, and 30 nM DnaB₆(DnaC)₆ at 10 μl/min. Reactions were carried out 31°C, maintained by an electrically heated chamber (Okolab, Burlingame, CA).
Double-stranded DNA was visualized in real time by staining it with 150 nM SYTOX orange (Invitrogen) excited by a 568 nm laser (Coherent, Santa Clara, CA; Sapphire 568–200 CW) at 150 μW/cm. The red and green Pol III* were excited at 700 mW/cm² with 647 nm (Coherent, Obis 647–100 CW) and 488 nm (Coherent, Sapphire 488–200 CW) lasers, respectively (Figure 1—figure supplement 1). The signals were separated via dichroic mirrors and appropriate filter sets (Chroma, Bellows Falls, VT). Imaging was done with either an EMCCD (Photometics, Tucson, AZ; Evolve 512 Delta) or a sCMOS camera (Andor, UK; Zyla 4.2). The analysis was done with ImageJ using in-house built plugins. The rate of replication of a single molecule was obtained from its trajectory and calculated for each segment that has constant slope.
Conditions for the pre-assembly replication reactions were adapted from published methods (Tanner et al., 2011 (link); Georgescu et al., 2011 (link)). Solution 1 was prepared as 30 nM DnaB₆(DnaC)₆, 1.5 nM biotinylated ds M13 substrate and 1 mM ATP in replication buffer. This was incubated at 37°C for 3 min. Solution 2 contained 60 μM dCTP and dGTP, 6.7 nM red Pol III*, and 74 nM β₂ in replication buffer (without dATP and dTTP). Solution 2 was added to an equal volume of solution 1 and incubated for 6 min at 37°C. This was then loaded onto the flow cell at 100 μl/min for 1 min and then 10 μl/min for 10 min. The flow cell was washed with replication buffer containing 60 μM dCTP and dGTP. Replication was finally initiated by flowing in the imaging buffer containing 50 nM β₂, 300 nM DnaG and 250 nM SSB₄ at 10 μl/min.

HUVECs (ATCC, CRL-1730) were cultured in growth media (EGM-2 Endothelial Cell Growth Medium-2 BulletKit, Lonza) containing growth factors IGF1, FGF2, EGF, and VEGFA according to the manufacturer’s instructions, unless otherwise stated. Cells (5 × 10³ per 96-well) were stimulated with 1,000 U/mL human IFN-α (1100-1, PBL Assay Science), and/or 100 ng/mL IL-22 (200-22, PeproTech) for 8 hours for gene expression analyses or for 5 days for survival and proliferation analyses. For survival experiments, cells were stained with SYTO 13/SytoX ORANGE (S34854, S11368, Invitrogen) and analyzed by flow cytometry. Live cells were counted as total Syto single-positive cells per condition. For proliferation experiments, cells were fixed, permeabilized (FIX & PERM Cell Fixation & Permeabilization Kit, Thermo Fisher Scientific), stained for KI67, and analyzed by flow cytometry.