FIGURE 3.

Primer elongation capability of DNA polymerase ϵ is enhanced by Tim-Tipin and Tim. A, elongation activity of DNA polymerase ϵ was measured on a 40-/80-mer as the primer/template, as described under “Experimental Procedures.” A fixed amount of DNA polymerase ϵ (0.2 nm) was assayed alone (lane 2) or in the presence of increasing concentrations of recombinant Tim or Tim-Tipin complex (12.5, 25, 50, 100, 200, 400, 700 nm; lanes 3–9 and lanes 11–17, respectively). Control assays were carried out which contained only Tim or Tim-Tipin (at 700 nm) in the absence of DNA polymerase ϵ (lanes 10 and 18, respectively). All of the reactions, including the blank sample (lane 1), were stopped and loaded on a 12% polyacrylamide/bisacrylamide (19:1) gel containing 7 m urea in 0.5× TBE buffer. The electrophoretic run was carried out in the same buffer at 30 watts. B, the radioactivity of the elongated products was quantified using the ImageQuant software (GE Healthcare). The intensity of the signal in each lane was normalized to the value calculated for the reaction carried out by DNA polymerase ϵ alone. In the plot average values are reported with error bars from three independent experiments carried out in the presence of Tim (squares) or Tim-Tipin (circles).