Figure 4.

Loading of ³²P-labeled hPCNA and mutant PCNAs onto singly nicked pBS circular duplex DNA. (A) ATP-dependent loading. Reactions were carried out as described in Materials and Methods with 1–2 pmol of ³²P-PCNA trimer (1–1.5 × 10³ cpm/fmol of trimer) and varying amounts of RFC as indicated. The total amount of ³²P-PCNA-DNA complex eluted in the excluded volume at each RFC concentration added was determined and is reported above as PCNA loaded. In the absence of RFC, <2 fmol of PCNA was detected in the excluded volume. (B) Loading in the presence of nonhydrolyzable ATP analogues. Reactions were carried out as described in A.