FIGURE 8.

Physical and functional interaction of purified Tim 5 with DNA polymerase ϵ. A, immunoprecipitation experiments were carried out on a mixture of purified DNA polymerase ϵ, full-length Tim, or Tim 5 with α-Tim antibodies, previously immobilized on protein A-agarose, as indicated under “Experimental Procedures.” DNA polymerase ϵ (0.5 μg), full-length Tim (0.35 μg) and Tim 5 (0.21 μg), and bound proteins (lanes 1–8) were analyzed by Western blotting using α-FLAG antibodies. B, the elongation capability of a fixed amount of DNA polymerase ϵ (0.2 nm) alone (lane 2) or in the presence of increasing concentrations of purified Tim 5 (12.5, 25, 50, 100, 200, 400, 700 nm, lanes 4–10, respectively) or Tim-Tipin (700 nm, lane 11) was measured on a 40-/80-mer substrate, as indicated under “Experimental Procedures.” Control assays were carried out with Tim 5 alone (700 nm, lane 3).