P-labeled 16-mer primer and a 30-mer template (sequences given above) were annealed at a molar ratio of 1:1 to detect DNA polymerase activity (1
P-labeled 16-mer primer, a downstream oligomer (5′-AAGATGCTGACGAG
), and the 30-mer template were used at a molar ratio of 1:5:2 for the nicked substrate. Singly primed DNA templates were prepared by annealing the 5′-32
P-labeled 24-mer primer to single-stranded circular M13mp18GTGx DNA at a molar ratio of 2:1 to detect processivity. To measure strand displacement, substrates were prepared by mixing the 5′-32
P-labeled 24-mer primer, a downstream 60-mer oligomer with a 5′-phosphoryl, and single-stranded M13mp18GTGx at a molar ratio of 1:5:2. Primer templates were heated for 5 min at 65°C and cooled down slowly for annealing. Reaction mixtures (10 µl) in buffer A [20 mM Tris–HCl pH 8.8, 4% glycerol, 2 mM dithiothreitol (DTT), 80 µg/ml bovine serum albumin (BSA), 8 mM Mg acetate], 100 µM of each dNTP, 30 nM of the primer-template (unless otherwise indicated), and the indicated amount of POLN derivatives. Mixtures with POLQ (10 µl) contained buffer A and 0.1 mM EDTA. RB69 gp43 reaction mixtures (10 µl) contained 10 mM Tris–HCl pH 7.9, 50 mM NaCl, 1 mM DTT, 200 µg/ml BSA, 10 mM MgCl2
and 100 µM of each dNTP. After incubation at 37°C for 10 min (unless otherwise indicated), reactions were terminated by adding 10 µl of formamide stop buffer and boiling at 95°C for 3 min. Products were electrophoresed on a denaturing 20% polyacrylamide–7 M urea gel and exposed to BioMax MS film or analyzed with a Fuji FLA3000 Phosphor Imager. For translesion synthesis, the same amounts of templates containing specific lesions were used. For steady-state kinetics, 1 pmol of primer-template (100 nM) was used and the procedure was as previously described (12
). This procedure utilizes extensive titration of deoxynucleotide concentration (0.1, 1, 5, 10, 50, 100, 200, 500 and 1000 µM) and four time points (0, 2.5, 5 and 10 min), producing results valid for a wide range of enzyme concentrations. Vmax
were determined from a Hanes–Woolf plot of [dNTP]/velocity versus [dNTP]. The nucleotide misincorporation ratio, finc
was determined by dividing (kcat
. Less than 10% of the primers were extended under steady-state conditions, ensuring single hit conditions. In the steady state, Vmax
values were proportional to enzyme concentration (data not shown). Here, kcat
was presented by utilizing the equation kcat
(mol of primer-template)]/ [(mol of polymerase) min].
To test sensitivity to ddNTP, ddTTP (2′,3′-dideoxythymidine-5′-triphosphate, Amersham, Piscataway, NJ, USA) and a poly(dA)-oligo(dT)10:1 template were used. Reaction mixtures (25 µl) in buffer A contained 8 µg/ml of poly(dA)-oligo(dT)10:1, 10 µM of dTTP, the indicated amount of ddTTP, 1 µCi of [α-32P] dTTP, 23 nM of WT (POLN) or delP, or 115 nM of Y682F, or 1 pM of Kf (exo−). After incubation at 37°C for 20 min, reactions were stopped by adding 25 µl of 40 mM EDTA and placed on ice. A 10 µl aliquot of each mixture was spotted onto DE81 paper (Whatman), and washed three times with 0.5 M Na2HPO4 for 5 min and twice with ethanol. The paper was dried and radioactivity was quantified with a Fuji Phosphor Imager.