Primer Extension Assay for DNA Polymerase

Primer extension reactions were performed at 30°C in polymerisation buffer (25 mM Tris-HCl pH 7.5, 8 mM Magnesium Acetate, 5 mM Potassium Glutamate, 5% Glycerol). Purified proteins used in the primer extension assays were purified as previously described (Devbhandari and Remus, 2020 (link)). DNA template for the assay was generated by annealing a primer (5′-CCCAGTCACGACGTTGTAAAACG-3′) to M13mp18 single-stranded DNA (New England Biolabs, N4040S). The assay was initiated by incubation of 1 nM of DNA template with 1mM ATP, 1mM DTT, 80 μM dATP, 80 μM dGTP, 80 μM dCTP and 400 nM of RPA for 5 min. PCNA and RFC were then added to 70 nM and 4 nM, respectively, and incubation was continued for 5 min. Then, 33 nM of α−³²P-dATP (3000 Ci per mmol) and 4 nM of either Pol δ^WT or Pol δ^D941Y was added to the reaction, resulting in a primer extension by nine base pairs (due to lack of dTTP). After 5 min, 80 μM dTTP were added to the mix for synchronous primer extension. Equal volume aliquots of this reaction (18 μl) were removed from the master reaction (100 μl) at indicated times and stopped by adding EDTA and SDS to final concentrations of 40 mM and 0.25%, respectively. Products were fractionated on a 0.8% alkaline agarose gel (30 mM NaOH and 2 mM EDTA), dried and imaged using Typhoon FLA 7000. Quantification of the gel images was performed using ImageJ.