Pol IV DNA Polymerase Assay.

The DNA template used to measure the DNA polymerase activity of Pol IV and mutants was prepared by annealing a ³²P-labeled 25-mer oligonucleotide labeled with ³²P at the 5′-end, to circular M13 single-stranded DNA. Reaction mixtures (20 μL) containing 20 mM Tris⋅HCl [pH 7.5], 8 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA, 40 μg/mL bovine serum albumin (BSA), 4% glycerol, 200 μM each dNTPs, 1 μM SSB, 8 nM primed template [5′-³²P-CGACGTTGTAAAACGACGGCCAGTG-3′′ annealed to M13 Ophrys single-stranded DNA (8.6 knt) (52)], and the indicated concentrations of Pol IV were incubated at 37 °C for 5 min. Reactions were stopped by the addition of a twofold volume of loading buffer (50 mM EDTA, 1 mg/mL xylene cyanol FF, and 1 mg/mL bromophenol blue in formamide). Samples were denatured at 95 °C for 5 min and the DNA products analyzed by denaturing gel electrophoresis (12% polyacrylamide [acrylamide:bisacrylamide 29:1], 8 M urea, and 1× Tris-borate-EDTA buffer [89 mM Tris base, 2 mM EDTA, and 89 mM boric acid]) at 8 V/cm for 2 h at room temperature. The gel was dried on Whatman paper filter (GE Healthcare Life Sciences) at 80 °C under vacuum for 2 h. Radiolabeled products were scanned with a STORM 860 Phosphorimager (Molecular Dynamics). The intensity of unextended primer (³²P-labeled 25-mer oligonucleotide) was quantified by ImageQuant 5.2 software.