All proteins used herein were purified from Escherichia coli K-12 strains (BL21(DE3) and DH5α) as described below. The replication template, M13Ophrys, was purified from E. coli XL2-MRFʹ as described below. The ssDNA calibration standard, λGap, was generated from λKytos (), which was purified from E. coli LE392.

Method Details

Microscopy Amitani et al., 2010 Amitani I.

Liu B.

Dombrowski C.C.

Baskin R.J.

Kowalczykowski S.C. Watching individual proteins acting on single molecules of DNA. Forget et al., 2013 Forget A.L.

Dombrowski C.C.

Amitani I.

Kowalczykowski S.C. Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. 50 flap, anchored to the surface via biotin-streptavidin linkage. The microscope was fitted with a CRISP autofocus system (ASI Instruments). The temperature of the flow-cell was maintained at 37°C throughout by a heated jacket fitted around the objective fed from a thermostatically controlled water bath. Images were captured using a DU-897E iXon EMCCD camera (Andor, 100 ms exposure), with an effective pixel size of 162.9 nm at 100 × magnification. Microscopy was performed on an Eclipse TE2000-U, inverted TIRF microscope (Nikon), using a CFI Plan Apo TIRF 100×, 1.49 numerical aperture, oil-immersed objective, and 488 nm and 561 nm lasers, as previously described (). Reactions were performed in single-use flow-channels, formed as described below. The replication template was an 8.6 kb Form II derivative of M13mp7 bearing a 5′ biotinylated dTflap, anchored to the surface via biotin-streptavidin linkage. The microscope was fitted with a CRISP autofocus system (ASI Instruments). The temperature of the flow-cell was maintained at 37°C throughout by a heated jacket fitted around the objective fed from a thermostatically controlled water bath. Images were captured using a DU-897E iXon EMCCD camera (Andor, 100 ms exposure), with an effective pixel size of 162.9 nm at 100 × magnification.

Coverslip preparation 2 SO 4 (conc.) to 1 part 30% (v/v) H 2 O 2 ), followed by submerging four times in MilliQ water and 30 min methanolic KOH (∼1.3 M) treatment with sonication. After submerging a further four times in MilliQ water, coverslips were submerged twice for 10 min in CMOS-grade acetone (JT Baker), the second time with sonication. Coverslips were functionalized with primary amine groups using (3-aminopropyltriethoxy)silane (Sigma; 2% (v/v) in acetone) for 5 min with agitation, submerged an additional four times in MilliQ water, and baked at 120°C for ∼30 min to cure the silane. After cooling, coverslips were PEGylated by applying a viscous mixture of PEG and biotin-PEG to one side of the coverslip; a second coverslip was placed on top of the first to make a sandwich, and a third non-functionalized coverslip placed along the edge of the sandwich to aid prising apart of the PEGylated coverslips. The outside faces of the sandwich were scribed to aid identification of the PEGylated side ( Tanner and van Oijen, 2009 Tanner N.A.

van Oijen A.M. Single-molecule observation of prokaryotic DNA replication. 3 (pH 8.4) with a mix of 1:50 biotin-PEG-NHS ester to mPEG-NHS ester (Nanocs); total final PEG concentration ∼15% (w/v). Following 4-5 rinses using a jet of MilliQ water, coverslips were dried under a stream of N 2 gas and stored in the dark, under vacuum, at room temperature, for up to two weeks before use. Coverslips (FisherFinest #1, 22 mm square) were subjected to a 30 min piranha clean (3 parts HSO(conc.) to 1 part 30% (v/v) H), followed by submerging four times in MilliQ water and 30 min methanolic KOH (∼1.3 M) treatment with sonication. After submerging a further four times in MilliQ water, coverslips were submerged twice for 10 min in CMOS-grade acetone (JT Baker), the second time with sonication. Coverslips were functionalized with primary amine groups using (3-aminopropyltriethoxy)silane (Sigma; 2% (v/v) in acetone) for 5 min with agitation, submerged an additional four times in MilliQ water, and baked at 120°C for ∼30 min to cure the silane. After cooling, coverslips were PEGylated by applying a viscous mixture of PEG and biotin-PEG to one side of the coverslip; a second coverslip was placed on top of the first to make a sandwich, and a third non-functionalized coverslip placed along the edge of the sandwich to aid prising apart of the PEGylated coverslips. The outside faces of the sandwich were scribed to aid identification of the PEGylated side (). PEGylation was at room temperature in the dark for ∼3 hr, in 100 mM NaHCO(pH 8.4) with a mix of 1:50 biotin-PEG-NHS ester to mPEG-NHS ester (Nanocs); total final PEG concentration ∼15% (w/v). Following 4-5 rinses using a jet of MilliQ water, coverslips were dried under a stream of Ngas and stored in the dark, under vacuum, at room temperature, for up to two weeks before use.

Flow-cell assembly 2 laser cutter followed by drilling ( Forget et al., 2013 Forget A.L.

Dombrowski C.C.

Amitani I.

Kowalczykowski S.C. Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. Each flow-cell consisted of three independent single-use channels, each of which had its own inlet and outlet port. Holes of ∼1 mm diameter, 10 mm apart, were etched in standard 25 × 75 mm uncoated glass microscope slides with an Epilog COlaser cutter followed by drilling (). Slides were cleaned by immersion in 2% (v/v) Hellmanex III solution overnight followed by sonication for 30 min in methanolic KOH (∼1.3 M). Inlet and outlet ports, made of ∼1 cm long PEEK tubing (Upchurch Scientific, #1532) were sharpened using a rotary sander, inserted into the cut holes of the microscope slides, and glued in place using a five-minute epoxy (Loctite). Once the epoxy had set, the sharpened ends of the PEEK tubing were trimmed with a razor blade. Three 2.5 × 12.5 mm channels were cut using a Craft Robocutter (GraphTec) in 19 × 19 mm squares of double-sided tape (3M #9437; thickness 51 μm), and flow-cells were assembled with double-sided tape sandwiched between slide and coverslip. The resulting flow-channels were 2.5 × 12.5 × 0.051 mm (∼1.6 μl). For live reactions, the channels were cut with dimensions of 1.25 × 12.5 × 0.051 mm, resulting in a flow-cell volume of ∼0.8 μl.

Replication template Bell et al., 2012 Bell J.C.

Plank J.L.

Dombrowski C.C.

Kowalczykowski S.C. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA. 2 PO 4 , 36 mM K 2 HPO 4 , and 0.5% (v/v) glycerol), containing 100 μg/ml ampicillin was inoculated with a single colony from the agar plate and grown overnight at 37°C with shaking at 200 rpm. E. coli cells were pelleted by centrifugation at 4,000 x g for 30 min and the supernatant containing phage retained. Phage was precipitated by the addition of 0.5 M NaCl, 5% (w/v) PEG-8000 (final concentrations) to the supernatant on ice for 2 hr, pelleted by centrifugation at 4,000 x g for 30 min, and resuspended in ice-cold buffer containing 25 mM Tris-HCl (pH 7.5 at 4°C), 10 mM Mg(OAc) 2 , 100 mM NaCl, and 50% (v/v) glycerol. The phage stock was stored at –20°C until use. An 8.6-kb derivative of M13mp7 circular ssDNA bearing the attB integration site for ϕC31 integrase (M13Ophrys) and an ampicillin resistance gene was used as template (). M13Ophrys was purified as follows. First, a phage stock was prepared by transforming the double-stranded form of the vector into DH5α and plating on LB agar containing 100 μg/ml ampicillin at 37°C. The following day, 25 mL of ‘GB’ medium (LB containing 8.5 mM KHPO, 36 mM KHPO, and 0.5% (v/v) glycerol), containing 100 μg/ml ampicillin was inoculated with a single colony from the agar plate and grown overnight at 37°C with shaking at 200 rpm. E. coli cells were pelleted by centrifugation at 4,000 x g for 30 min and the supernatant containing phage retained. Phage was precipitated by the addition of 0.5 M NaCl, 5% (w/v) PEG-8000 (final concentrations) to the supernatant on ice for 2 hr, pelleted by centrifugation at 4,000 x g for 30 min, and resuspended in ice-cold buffer containing 25 mM Tris-HCl (pH 7.5 at 4°C), 10 mM Mg(OAc), 100 mM NaCl, and 50% (v/v) glycerol. The phage stock was stored at –20°C until use. To generate the circular ssDNA form of M13Ophrys, E. coli XL2 Blue-MRFʹ was streaked on LB agar containing 34 μg/ml chloramphenicol and grown overnight at 37°C. In the morning, a single colony was inoculated into 3 mL of GB medium (above) containing 34 μg/ml chloramphenicol in a test tube, and incubated at 37°C with shaking at 200 rpm. After ∼4 hr, once the culture had become turbid, 100 μl of the phage stock generated above was added and growth continued at 37°C for ∼2 hr. 100 mL of GB medium (above) containing 34 μg/ml chloramphenicol and 50 μg/ml ampicillin was inoculated with 100 μl of the phage-infected culture, and growth continued overnight at 37°C. 50 mL of the culture was pelleted the following morning by centrifugation at 4,000 x g; the supernatant containing phage was retained and pelleted as described above. Circular M13 ssDNA was purified using a QIAGEN maxiprep kit, using buffers supplied by the manufacturer, and with the protocol modified as follows: phage pellets were resuspended in 10 mL Buffer P1; 10 mL Buffer P2 was added, mixed gently, and incubated for 5 min to lyse the phage. 10 mL Buffer P3 was added to neutralize the sample and precipitate the protein, SDS and DNA for 10 min, followed by clarification by centrifugation at 4,000 x g for 30 min. The clarified supernatant was further filtered through a Kimwipe using a 50 mL plastic syringe. A single QIAGEN tip-500 column was equilibrated with 15 mL Buffer QBT. The clarified lysate was added to the column and allowed to enter the resin bed. The column was washed with 5 mL of ssDNA wash buffer (50 mM MOPS (pH 7.0), 900 mM NaCl, 15% (v/v) isopropanol), then twice further, each time with 30 mL ssDNA wash buffer. Circular DNA was eluted with 20 mL of 50 mM MOPS (pH 7.0), 1 M NaCl, 4 M deionized urea, 15% isopropanol; precipitated with the addition of 14 mL of isopropanol; and pelleted by centrifugation at 4,000 x g for 60 min. The DNA pellet was washed twice with 10 mL of room temperature 70% (v/v) EtOH, and dissolved overnight at 4°C in 100 μl TE buffer (10 mM Tris-Cl (pH 8.0 at 4°C), 1 mM EDTA). The purity and integrity of the substrate was verified by 0.8% alkaline and neutral agarose gel electrophoresis. To generate the rolling-circle template, oligonucleotide oJEG38 (5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT AATTCGTAATCATGGTCATAGCTGTTTCCT-3′), bearing a 5′ biotin-triethylene glycol moiety and 50-nt flap, was synthesized (Integrated DNA Technologies) and purified by urea-PAGE and avidin-agarose. The oligo was annealed to M13Ophrys via 30 nt of complementarity and extended using T7 DNA polymerase (NEB) in the presence of 200 μM dNTPs to create a biotin-tailed Form II template. Extension of the primer to generate a nicked circle was monitored by 0.8% alkaline and neutral agarose gel electrophoresis of the products. Products were purified by two rounds of phenol-chloroform extraction followed by ethanol precipitation.

Recombinant proteins ∗ were purified as described ( Hiasa and Marians, 1996 Hiasa H.

Marians K.J. Two distinct modes of strand unlinking during theta-type DNA replication. Marceau et al., 2011 Marceau A.H.

Bahng S.

Massoni S.C.

George N.P.

Sandler S.J.

Marians K.J.

Keck J.L. Structure of the SSB-DNA polymerase III interface and its role in DNA replication. Marians, 1995 Marians K.J. Phi X174-type primosomal proteins: purification and assay. Xu and Marians, 2000 Xu L.

Marians K.J. Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function. 6 -tags, were purified as follows. Primase was overexpressed in E. coli BL21(DE3) by adding 1 mM IPTG to the culture at OD 600 ∼0.8 for 4 hr at 37°C. Cells were lysed by passage twice through a French press (12,000 psi) in 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 150 mM NaCl, 20 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20 mM spermidine, and debris removed by centrifugation at 44,000 × g for 20 min. The pH of the lysate was adjusted to 8.0 using solid Tris base. Following lysis, genomic DNA was precipitated and removed by dropwise addition of a 10% (v/v) solution of Polymin P (pH 8.0) to 0.04% (v/v) and centrifugation at 30,000 × g for 20 min. Protein in the resulting supernatant was precipitated with 50% saturated ammonium sulfate and centrifuged as above. The pellet was dissolved in ∼50 mL of 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 500 mM NaCl, 10 mM imidazole and dialyzed for 2 hr against the same buffer in a volume of 4 L. The dialysate was applied to a 5 mL HisTrap column (GE) and protein eluted in a gradient of 0-500 mM imidazole over ten column volumes. Pooled fractions (∼30 mg) were diluted to 50 mL by 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 10 mM NaCl; 1.4 mg His-tagged TEV protease was added for cleavage overnight at 4°C. The cleaved protein was reapplied to the HisTrap column as above, and untagged primase collected in the flow-through. Cleaved mutant primase was dialyzed ∼2 hr against 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 10 mM NaCl, 1 mM EDTA, 5 mM DTT (Buffer A) and diluted ∼3-fold for application to a 5 mL HiTrap heparin column equilibrated with the same buffer. The flow-through was reapplied twice to the column. Protein was eluted in a 10-600 mM NaCl gradient over ten column volumes; DnaG eluted at ∼125 mM NaCl. Approximately 9 mg total protein was obtained. The protein was dialyzed against Buffer A, applied to a MonoQ (10/10) column equilibrated with Buffer A, and eluted with a 10-500 mM NaCl gradient over ten column volumes. The pure fractions (6.6 mg protein) were dialyzed against 50 mM Tris-Cl (pH 7.5 at 4°C), 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 38% (v/v) glycerol, and snap-frozen in liquid nitrogen for storage at –80°C. The mutant primase preparations contained no detectable ssDNA or dsDNA nuclease activity. Untagged E. coli DnaB, DnaC810, wild-type primase (DnaG), β (DnaN), SSB and Pol IIIwere purified as described (). Mutant E. coli primases (D269A, D269Q) bearing N-terminal TEV-cleavable His-tags, were purified as follows. Primase was overexpressed in E. coli BL21(DE3) by adding 1 mM IPTG to the culture at OD∼0.8 for 4 hr at 37°C. Cells were lysed by passage twice through a French press (12,000 psi) in 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 150 mM NaCl, 20 mM EDTA, 1 mM DTT, 1 mM PMSF, and 20 mM spermidine, and debris removed by centrifugation at 44,000 × g for 20 min. The pH of the lysate was adjusted to 8.0 using solid Tris base. Following lysis, genomic DNA was precipitated and removed by dropwise addition of a 10% (v/v) solution of Polymin P (pH 8.0) to 0.04% (v/v) and centrifugation at 30,000 × g for 20 min. Protein in the resulting supernatant was precipitated with 50% saturated ammonium sulfate and centrifuged as above. The pellet was dissolved in ∼50 mL of 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 500 mM NaCl, 10 mM imidazole and dialyzed for 2 hr against the same buffer in a volume of 4 L. The dialysate was applied to a 5 mL HisTrap column (GE) and protein eluted in a gradient of 0-500 mM imidazole over ten column volumes. Pooled fractions (∼30 mg) were diluted to 50 mL by 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 10 mM NaCl; 1.4 mg His-tagged TEV protease was added for cleavage overnight at 4°C. The cleaved protein was reapplied to the HisTrap column as above, and untagged primase collected in the flow-through. Cleaved mutant primase was dialyzed ∼2 hr against 50 mM Tris-Cl (pH 7.5 at 4°C), 10% (v/v) glycerol, 10 mM NaCl, 1 mM EDTA, 5 mM DTT (Buffer A) and diluted ∼3-fold for application to a 5 mL HiTrap heparin column equilibrated with the same buffer. The flow-through was reapplied twice to the column. Protein was eluted in a 10-600 mM NaCl gradient over ten column volumes; DnaG eluted at ∼125 mM NaCl. Approximately 9 mg total protein was obtained. The protein was dialyzed against Buffer A, applied to a MonoQ (10/10) column equilibrated with Buffer A, and eluted with a 10-500 mM NaCl gradient over ten column volumes. The pure fractions (6.6 mg protein) were dialyzed against 50 mM Tris-Cl (pH 7.5 at 4°C), 50 mM NaCl, 1 mM EDTA, 5 mM DTT, 38% (v/v) glycerol, and snap-frozen in liquid nitrogen for storage at –80°C. The mutant primase preparations contained no detectable ssDNA or dsDNA nuclease activity.

Preparation of flow-cells for imaging Single-molecule imaging buffer (SMB) contained: 50 mM HEPES-KOH (pH 8.0), 10 mM magnesium acetate, 25 mM KCl, 15% (w/v) sucrose. DTT (50 mM) was added in powdered form fresh to the buffer on the day of use; the buffer was passed through a 0.22-μm syringe-driven filter and degassed for ∼4 hr. Each flow-channel was prepared immediately before use by pipetting in the following sequence: 100 μL SMB; 100 μL SMB containing 0.1 mg/ml streptavidin (Promega; ∼21°C, 2 min); 100 μL SMB. The flow-cell was then connected to the injection system using ∼5 mm pieces of PharMed tubing (Bio-Rad) to connect the PEEK tubing to the inlet and outlet ports. All surfaces, including connecting tubing, were blocked against nonspecific binding by flowing 1 mL of SMB containing 1% (w/v) blocking reagent (Roche; cat. 11096176001) through the flow-cell at 10 ml⋅h-1. Blocking reagent was removed by flowing another 1 mL of SMB through the system at 10 ml⋅h-1. The replication template was adsorbed to the surface by introducing SMB containing ∼10 pM (molecules) replication template and 75 nM SYTOX Orange into the flow-cell. The density of DNA molecules could be adjusted by a 10-fold concentration of template (from 10 to ∼100 pM) and time (from 1 to ∼10 min) without buffer flow; for end-point reactions, a lower density of molecules was preferred so that molecules would not overlap when elongated by replication.

Rolling-circle replication reactions The overall experimental scheme was to first attach an 8.6 kb template via a 5′ biotinylated tail to the surface (‘anchor’). Next, idling replisomes were formed by introducing DnaB, DnaC810, Pol III∗, β, three out of the four dNTPs, and all rNTPs. Following a wash to remove unbound proteins, replication was initiated by adding dTTP and flow-stretching the DNA. SSB, β and primase were present in flow. Specifically, following adsorption of DNA to the surface and the removal of excess DNA once the desired density of template had been achieved, the syringe, lines and flow-cell were equilibrated with wash buffer (‘WB’; SMB (as above) plus 1 mM ATP, 200 μM each CTP, GTP and UTP, 40 μM each dATP, dCTP and dGTP [no dTTP], and 75 nM SYTOX Orange). On the day of use, replication proteins were diluted to fifteen times the required concentration from stock in a buffer containing 50 mM Tris-Cl (pH 7.5), 10 mM beta-mercaptoethanol, 1 mM EDTA, 100 mM NaCl, 500 μg/ml BSA, and 20% (v/v) glycerol (protein diluent, ‘PD’). Reactions were performed in two stages: a pre-incubation “bind” mix (50 μl) contained 60 nM DnaB (as hexamer), 380 nM DnaC810, 20 nM Pol III∗, 30 nM DnaN (β, as dimer), and 10 μg/ml BSA in WB (with additionally one-fifth equivalent of PD). Primase was not added to the pre-incubation mix. Flow-cells were washed with 25 μL WB (∼90 s, 1,000 μl⋅h-1), after which reactions were initiated with a “start” mix (60 μL for end-point reactions; 150 μL for live reactions), containing 320 nM primase, 250 nM SSB (as tetramer), 30 nM DnaN (β, as dimer), 40 μM dTTP (in addition to the other three dNTPs), and 10 μg/ml BSA in WB (with additionally one-fifth equivalent of PD). Pre-incubation, wash, and start mix were injected in-line into the flow-cell using a system of injection loops driven by a syringe pump. Flow was maintained at 1,000 μl⋅h-1 during the pre-incubation and wash stages, and was altered as appropriate during the reaction (below). The volume required to initiate the reaction was determined empirically by injecting fluorescent dye into the flow-cell. For dropout and titration experiments, the concentration of protein was varied as appropriate (see main text).

End-point replication reactions -1 without constant laser illumination; reactions were only periodically illuminated with the 561 nm laser to check the progress of the reaction. At 10 min, reactions were quenched by injection of 50 μL SMB containing 75 nM SYTOX Orange, 1 mM AMP-PNP, 40 μM each dATP, dCTP and dGTP and ddTTP, at 4,000 μl⋅h-1. The flow-cell and lines were washed and equilibrated with SMB lacking magnesium acetate, plus 15 nM SYTOX Orange (“end-point imaging buffer”). The removal of Mg2+ ion (i) prevented further DNA synthesis; (ii) improved the signal-to-noise ratio of fluorescence intensity detection, as the affinity of SYTOX Orange stain for dsDNA is higher in the absence of divalent cation; and (iii) relieved the compaction of ssDNA⋅SSB, improving the resolution of regions of any possible gaps on the lagging strand (-1 for visualization, and an additional 20-50 frames recorded. Between 40 and 200 fields were recorded for each flow-channel by imaging in a serpentine path to avoid field duplication. For each dataset, a ‘flat’ image was recorded by defocusing 1-2 μm from the surface into bulk solution and taking an average of 8-10 points in the flow-channel. Each image for that dataset was then normalized by dividing the intensity values by the ‘flat’ image values. For end-point reactions, flow was reduced to 100 μl⋅hwithout constant laser illumination; reactions were only periodically illuminated with the 561 nm laser to check the progress of the reaction. At 10 min, reactions were quenched by injection of 50 μL SMB containing 75 nM SYTOX Orange, 1 mM AMP-PNP, 40 μM each dATP, dCTP and dGTP and ddTTP, at 4,000 μl⋅h. The flow-cell and lines were washed and equilibrated with SMB lacking magnesium acetate, plus 15 nM SYTOX Orange (“end-point imaging buffer”). The removal of Mgion (i) prevented further DNA synthesis; (ii) improved the signal-to-noise ratio of fluorescence intensity detection, as the affinity of SYTOX Orange stain for dsDNA is higher in the absence of divalent cation; and (iii) relieved the compaction of ssDNA⋅SSB, improving the resolution of regions of any possible gaps on the lagging strand ( Figure S1 F). Where necessary (main text), the flow was shut off for ≥ 20 s and the molecules allowed to recoil. Between 20 and 50 frames at 100 ms exposure were recorded. All end-products were extended at a flow-rate of 4,000 μl⋅hfor visualization, and an additional 20-50 frames recorded. Between 40 and 200 fields were recorded for each flow-channel by imaging in a serpentine path to avoid field duplication. For each dataset, a ‘flat’ image was recorded by defocusing 1-2 μm from the surface into bulk solution and taking an average of 8-10 points in the flow-channel. Each image for that dataset was then normalized by dividing the intensity values by the ‘flat’ image values.

Live replication reactions Reactions were performed as described above, except in a flow-cell of half the width (∼0.8 μL volume) to permit double the linear flow-rate for a given volumetric flow-rate. Upon starting the reaction with a defined volume of the ‘start’ mix, the flow-rate was adjusted to 1,250 μl⋅h-1, the equivalent of 2,500 μl⋅h-1 in the end-point reactions above. Movies were recorded at a low level of laser exposure, determined empirically not to cause appreciable photocleavage to λ DNA, at a frame rate of 7.4 Hz.

Okazaki fragment end-labeling and imaging Reactions were performed exactly as for the end-point experiments above, except that at 10 min, 50 μL of SMB containing 1 M NaCl was injected into the flow-cell to remove bound proteins and SYTOX Orange. The high-salt buffer was immediately followed by 400 μL WB at 4,000 μl⋅h-1 without stopping the flow. Replication products were pulse-labeled by injecting 50 μL of a mix comprising: 30 mM Tris-Cl (pH 8.0 at 25°C), 10 mM magnesium acetate, 10 μg/ml BSA, 40 μM each of dATP, dCTP and dGTP, 20 μM digoxygenin-11-dUTP (alkali-stable, Roche), 26 μM NAD+, 3.3 U DNA Pol I (wild-type, full-length, NEB), 3.3 U E. coli DNA ligase (NEB), and 250 nM SSB (as tetramer), at a flow-rate of 500 μl⋅h-1 for 5 min. At 5 min, a further 50 μL SMB + 1 M NaCl was introduced to remove bound Pol I and ligase, immediately followed by a further wash of 400 μL WB at 4,000 μl⋅h-1, again without stopping flow. Sheep polyclonal anti-digoxigenin F(ab) fragments (Roche), labeled with Alexa Fluor 488 NHS ester (ThermoFisher Scientific) with an average of 3.6 dyes per molecule, were injected into the flow-cell in end-point imaging buffer, at ∼300 nM molecules; 250 nM SSB (tetramer) was also included to replenish any removed during the high-salt wash. Excess F(ab) fragments and SSB were washed out of the flow-cell and products imaged using the same buffer. There was no appreciable nonspecific binding of labeled F(ab) fragments to the surface. Alexa Fluor 488 and SYTOX Orange signals were imaged on two halves of the EMCCD using a Dual-View apparatus (Optical Insights) and bandpass filters specific to each dye (Chroma), illuminating the sample alternately with the 488 nm and 561 nm lasers. DNA was imaged under a flow-rate of 8,000 μl⋅h-1. The 5′ and 3′ locations of each OF were inferred from the patterns of 3′ terminus labeling and the positions of gaps (see Figure 5 A). To estimate the labeling efficiency, we determined the proportion of resolvable ssDNA⋅SSB gaps which had a detectable focus at their right-hand edge, i.e., the 3′ terminus of the right-hand OF ( Figure 5 A), which acted as an internal control ( Table S1 ).

Preparation of λ DNA with an ssDNA gap Thorpe and Smith, 1998 Thorpe H.M.

Smith M.C. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Bacteriophage M13mp7 ssDNA containing the attB recognition site was used to generate a 500 bp dsDNA fragment containing the ϕC31 attB at its center by PCR using the Phusion High Fidelity PCR Master Mix from NEB. The dsDNA product was heat denatured, and then annealed to the M13mp7 ssDNA derivative. The gapped λ DNA was generated by site-specific recombination between λKytos dsDNA and the annealed M13mp7 ssDNA containing the attB recognition site (M13Ophrys), using ϕC31 integrase. The ϕC31 integrase was purified from plasmid pHS62 ().