T7 express competent cells

The plasmid containing PolD and PolD H931A were transformed into T7 Express competent cells (New England Biolabs, Ipswich, MA). Two liters of cells were grown at 37°C until an OD₆₀₀ of 0.4 was reached, at which time the cells were induced with 0.4 mM (final concentration) of IPTG. Growth was continued for 3 h at 37°C and cells were harvested by centrifugation. The cell pellet was resuspended in Buffer A and lysed using a constant cell disruptor. The cell lysate was heated to 80°C for 20 min in a water bath and the lysate was clarified by centrifugation at 4,000 × g for 20 min. The clarified supernatant was loaded onto a HiPrep 16/10 DEAE column (GE Lifesciences, Pittsburg, PA, USA) and the flow-through was collected and loaded onto a 5 ml HisPrep FF column equilibrated with Buffer A. The polymerases were eluted off the column in fractions using Buffer B and an imidazole gradient from 50–250 mM. Fractions containing PolD were identified by SDS-PAGE analysis, followed by a primer extension activity assay to confirm the presence of active enzyme (described above). Active enzyme was pooled and dialyzed into 10 mM Tris–HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol, pH 7.4 and protein concentration was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).

Plasmids containing PolD and variant PolDs were transformed into T7 Express competent cells (New England Biolabs, Ipswich, MA, USA). A 25 ml culture was grown in LB supplemented with 0.1 mg/ml ampicilin at 37°C to an OD₆₀₀ of 0.6. Protein expression was induced with 0.2 mM (final) Isopropyl β-d-1-thiogalactopyranoside (IPTG). Growth was continued for 3.5 h at 37°C and cells were harvested by centrifugation at 3,800 rpm for 30 min. Cell pellets were resuspended in 150 ml of Buffer A (20 mM Tris–HCl at pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 20 mM Imidazole) and lysed using a constant cell disruptor (Constant Systems LTD, Northants, UK). Cell lysates were heated to 80°C for 20 minutes and clarified by centrifugation at 30 000 × g for 30 min. The supernatant was filtered through a Whatman^TM grade filter paper (GE Healthcare, Buckinghamshire, UK) and loaded onto a 20 ml HisPrep^TM FF 16/10 column (GE Healthcare, Uppsala, Sweden) (pre-equilibrated with Buffer A) and eluted with Buffer B (20 mM Tris–HCl at pH 7.5, 300 mM NaCl, 0.1 mM EDTA, 500 mM Imidazole) with an imidazole gradient from 20 to 500 mM. Fractions were run on a 10–20% Tris–glycine gel (Invitrogen, Carlsbad, CA, USA) to confirm the presence PolD DP1 and DP2. Polymerase containing fractions were pooled and dialyzed in storage buffer (10 mM Tris–HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol, pH 7.4).