FIG. 4.

PCNA loading onto immobilized DNA is inhibited by soluble primed template DNA but not single-stranded DNA. A primed BTN3 template DNA fixed to the beads was first preincubated with 2 pmol of RPA for 5 min at room temperature and then mixed with 2.2 pmol of RFC, 20 pmol of PCNA trimer, and homopolymeric competitor DNA in the presence of 1 mM ATP (lanes 1 to 5) or ATPγS (lanes 6 to 10) for 30 min at room temperature. The beads were then washed with buffer A containing 0.2 mM ATP (lanes 1 to 5) or 0.2 mM ATPγS (lanes 6 to 10). The bound proteins were analyzed by Western blotting along with standards (S1 to S3) as described in the legend to Fig. 2. The competitor DNAs used were as follows: none (lanes 1 and 6), 250 pmol (in nucleotides) (lanes 2 and 7) or 500 pmol (lanes 3 and 8) of poly(dA)-oligo(dT) (pdA odT), and 250 pmol (lanes 4 and 9) or 500 pmol (lanes 5 and 10) of poly(dA) (pdA).