Typhoon 9400 scanner

To study the kinetics of the click reaction, 1D-SDS-PAGE analysis of TAMRA-labeled protein precipitates was performed. The final protein concentration was adjusted to 2 mg/mL. Samples were then heated for 10 min at 70 °C and 40 µg of protein were loaded. SDS-PAGE (12%) was carried out at 150 V. Gels were imaged on a Typhoon 9400 scanner (Amersham, ex 532 nm/em 580 nm). To normalize the fluorescence signal intensity on protein content/lane, Coomassie staining was performed and visualized on a ChemiDoc™ System (Biorad, Vienna, Austria).

The 30 µM (nucleotides) viral (+) strand of ΦX174 DNA were coated first with 3.3 µM RPA by incubation in SEB buffer (42 mM MOPS pH 7.2, 3 mM Mg acetate, 1 mM DTT, 20 mM KCl, 25 µg/mL BSA and 2.5 mM ATP) in a final volume of 12.5 µL at 37 °C for 10 min. Rad51 filament formation was initiated by adding 10 µM Rad51 and 15 µM WT or mutated Rad52, or storage buffer as control, at 37 °C for 20 min. Then, addition of 30 µM (nucleotides) of PstI-linearized ΦX174 dsDNA and 4 mM spermidine initiated the strand exchange reaction. After incubation at 37 °C for 90 min, samples were deproteinized by addition of 2 μL of 10 mg/mL proteinase K, 5% SDS solution at 37 °C for 10 min and analyzed by electrophoresis (0.8% agarose gels in 1 × TAE buffer). Standard reactions were done by adding Rad51 prior to RPA. Gels were stained with ethidium bromide and fluorescent signals were imaged with a Typhoon 9400 scanner (Amersham, GE Velizy-Villacoublay; France) and quantified with the Image J software (NIH, USA). The ratio of nicked circular product was calculated as the ratio between the sum of the linear dsDNA substrate and the nicked circular product.

Protein samples were boiled for 5 min in Laemmli loading buffer (except PAD1 samples, that remained unboiled), separated by SDS–PAGE (NuPAGE gel 4–12% Bis-Tris, Invitrogen) and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with lex = 457 nm and lem = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). After blocking with Odyssey Blocking buffer for at least 30 min at room temperature (RT), membranes were incubated with antibodies 1- to 3 hr at RT or overnight at 4°C under agitation in 2% BSA in TBS-T (0.1% Tween in TBS). The primary antibodies were used at the following dilutions: αPAD1 (1:1000); αBB2 (1:5, [Bastin et al., 1996 (link)]); αThioP (1:1000, Thio-phosphate ester [51-8], Abcam). After three washes in TBS-T, proteins were visualised by incubating the membrane for 1 hr at RT with a secondary antibody conjugated to a fluorescent dye diluted 1:5000 in 50%Odyssey Blocking buffer/50% TBS-T. Finally, membranes were scanned using a LI-COR Odyssey imager system.

Purified proteins were separated by SDS–PAGE (NuPAGE gel 4–12% Bis-Tris, Invitrogen) and visualized either by Coomassie staining or SYPRO Ruby Protein Gel Stain (Invitrogen) using a Typhoon 9400 scanner (Amersham Biosciences) with λ_ex = 457 nm and λ_em = 610 nm. Alternatively, proteins were separated by SDS–PAGE on NuPAGE 4–12% Bis-Tris gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF) membranes (Pierce). Proteins were revealed using the following antibodies at the indicated dilutions: i) polyclonal anti-MPK10 antibody, generated by rabbit immunization using recombinant MPK10 protein produced in E. coli transformed with pGEX-Strep3-MPK10 plasmid (Eurogentec), 1∶10,000; ii) anti-phospho-tyrosine antibody 4G10 Platinum from Millipore, 1∶1,000; and iii) secondary goat anti-rabbit-HRP and anti-mouse-HRP antibodies from Thermo Scientific, 1∶20,000. The visualization was performed on X-ray film (Roche) at various exposure times.

For the PrimPol DNA synthesis, the reaction mixture (10 μl) contained 10 mM Bis-Tris propane (pH 7.0), 40 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 200 μM dNTPs, 15 nM of hybridized 5′-TET-primer/template DNA, indicated amounts of purified PrimPol (WT or Δ56 mutant) and PolDIP2 (WT, truncated proteins, or mutants). After incubation for 10 min at 37°C, reactions were stopped by addition of 10 μl of formamide loading buffer (0.5% SDS, 25 mM EDTA, 95% v/v formamide and xylene-cyanol). For single hit experiments shown in Figure 1F and Supplementary Figure S2, the reaction composition was modified to include 50 nM hybridized primer/template DNA, 5 nM PrimPol WT, and indicated amounts of PolDIP2 fragments. Those reaction mixtures were incubated at 37°C for 30 min. DNA products of primer extension assays were separated on 10% polyacrylamide gels containing 7 M urea, and directly imaged using a Typhoon 9400 scanner (Amersham Bioscience). The images were quantified with ImageQuant TL 8.1 software (GE healthcare). The percentage of primer that was extended for >3 nt, >10 nt, or was fully extended, was calculated as the ratio between the signal of corresponding products and the signal of the 25-nt primer in the control reaction without protein. When used, the polymerisation termination probabilities at specific nucleotides were calculated as previously described (26 (link)).