FIGURE 5.

DNA polymerase activity of gp5 in the presence of trx. DNA synthesis was determined by measuring the amount of [³H]dTMP incorporated into DNA over time as described under “Experimental Procedures.” A, DNA polymerase activity on a homopolymer primer-template is shown. Reactions (200 μl) contained 50 mm Tris-HCl, pH 7.5, 5 mm DTT, 50 mm NaCl, 10 mm MgCl₂, 250 μm each of dGTP, dATP, dCTP, and [³H]dTTP (10 cpm/pmol), 200 nm dA₃₅₀/dT₂₅ substrate, 5 nm each of the indicated gp5s, and 250 nm trx. Reaction mixtures were incubated at 25 °C. Aliquots of 20 μl were removed at the indicated times, and the reaction was stopped by the addition of 25 mm EDTA. B, DNA polymerase activity on primed M13 ssDNA is shown. Reactions were similar to those described in A except that dA₃₅₀/dT₂₅ was replaced by 20 nm M13ss DNA primed with 24-nt primer. Additionally, reactions involving primed M13 ssDNA were incubated at 37 °C. ■, wt gp5; ○, gp5-C275S; ▴, gp5-C313S; ♦, gp5-C275S/C313S.