Primer extension reactions (25 μl) were based on method in (33 (link),34 (link)), utilizing purified human Pol δ (40 nM) in a holoenzyme complex with PCNA (40 nM), RFC (10 nM) and RPA (320 nM). RPA, PCNA and RFC were pre-mixed for 10 min on ice in the reaction buffer containing DNA substrate (82.5 ng M13-primer DNA/reaction) and dNTPs (200 μM), before adding Pol δ to start the reactions. Reactions were for 30 min at 37°C before adding 2 μl of STOP solution (50 mM Tris–HCl pH 8.0, 100 mM EDTA, 0.1% w/v SDS and 5 mg/ml of proteinase K). Cy5-labelled products after electrophoresis through a 0.8% agarose TBE gel were visualised using a Typhoon scanner, and unlabelled plasmid DNA was visualised by ethidium bromide staining and placing the gel on a UV transilluminator.
Primer extension reactions by isolated Pol δ complex (POLD1-D4) were in 20 μl containing substrate DNA (21 nt primer oligo annealed to a 70 nt oligo template, each at 15 nM), 10 mM MgCl2, 40 mM Tris–HCl pH7.5, 1 mM DTT, 0.2 mg/ml BSA, 50 mM NaCl and 200 μM of each dNTP. Primer extension assays using human DNA polymerase η and polymerase k were in buffer 40 mM Tris–HCl pH 8.0, 10 mM DTT, 0.25 mg/ml BSA, 60 mM KCl, 2.5% glycerol, 5 mM MgCl2, 200 μM dNTPs. Primer extension by E. coli polymerase III core (DnaE) and polymerase I were in buffer 10 mM magnesium acetate, 40 mM HEPES–NaOH pH 8.0, 0.1 mg/ml BSA, 10 mM DTT and 200 μM dNTPs. Unless stated otherwise, all polymerase primer extension assays were for 30 min at 37°C after adding DNA. Reactions were halted by adding 5 μl of stock STOP solution. Stopped reactions were mixed with loading dye (20% glycerol, 78% formamide and Orange G) for electrophoresis through 10% acrylamide (19:1 acrylamide: bis-acrylamide) TBE denaturing (8 M urea) gels, at 5 W for 3 h. Primer extension products were visualised via the Cy5-DNA end label using a Typhoon scanner, and files were quantified using ImageJ and Prism software. For primer extension reactions containing HelQ, proteins were pre-mixed in their storage buffers, in isolation from reaction buffer and DNA, and reactions commenced by adding buffer containing DNA and dNTPs.
Primer extension reactions by isolated Pol δ complex (POLD1-D4) were in 20 μl containing substrate DNA (21 nt primer oligo annealed to a 70 nt oligo template, each at 15 nM), 10 mM MgCl2, 40 mM Tris–HCl pH7.5, 1 mM DTT, 0.2 mg/ml BSA, 50 mM NaCl and 200 μM of each dNTP. Primer extension assays using human DNA polymerase η and polymerase k were in buffer 40 mM Tris–HCl pH 8.0, 10 mM DTT, 0.25 mg/ml BSA, 60 mM KCl, 2.5% glycerol, 5 mM MgCl2, 200 μM dNTPs. Primer extension by E. coli polymerase III core (DnaE) and polymerase I were in buffer 10 mM magnesium acetate, 40 mM HEPES–NaOH pH 8.0, 0.1 mg/ml BSA, 10 mM DTT and 200 μM dNTPs. Unless stated otherwise, all polymerase primer extension assays were for 30 min at 37°C after adding DNA. Reactions were halted by adding 5 μl of stock STOP solution. Stopped reactions were mixed with loading dye (20% glycerol, 78% formamide and Orange G) for electrophoresis through 10% acrylamide (19:1 acrylamide: bis-acrylamide) TBE denaturing (8 M urea) gels, at 5 W for 3 h. Primer extension products were visualised via the Cy5-DNA end label using a Typhoon scanner, and files were quantified using ImageJ and Prism software. For primer extension reactions containing HelQ, proteins were pre-mixed in their storage buffers, in isolation from reaction buffer and DNA, and reactions commenced by adding buffer containing DNA and dNTPs.
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