Figure 3.

Coupled in vitro transcription-translation reactions with plasmid DNA containing the coding sequence for the hRFC p140 and p38 subunits were carried out in the presence or absence of the p40·p37·p36 complex followed by immunoprecipitation using a polyclonal antibody against the hRFC p37 subunit. Because the C-terminal half of the p140 subunit (amino acids 555-1147) forms an hRFC complex that is 5–10 fold more active than that formed with the full-length p140 subunit, p140N555 was used in the experiments described here. The immunoprecipitated products were analyzed on SDS/9% polyacrylamide gels followed by autoradiography to visualize the ³⁵S-labeled p140 and p38, or assayed for their replication activities. (A) Reconstitution of hRFC from its five subunits. Reactions containing 20% of the input material (lanes 1, 3, 5, 7. 9, 11, 13, and 15) and the immunoprecipitates (lanes 2, 4, 6, 8, 10, 12, 14, and 16) were separated by SDS/9% polyacrylamide gels. The reactions contained wt or mutant hRFC subunits or subcomplexes as indicated. (B) Replication activities of mutant hRFCs. Mutant hRFCs, immunoprecipitated on protein A beads, were examined for their ability to elongate singly primed M13 DNA as described in Materials and Methods. Reactions shown in lanes 1–3 were carried out in the presence 70, 14, and 1.4 fmol of baculovirus RFC, respectively. Reactions shown in lanes 4–11 were carried out after immunoprecipitation of 2 μl of the reconstitution mixture as follows: lane 4, RFC reconstituted with wt p140, p38, and wt p40·p37·p36 complex; lane 5, the wt p40·p37·p36 complex only; lane 6, mutant p140 (p140K657A), wt p38, and wt p40·p37·p36 complex; lane 7, wt p140, mutant p38 (p38K48A), and wt p40·p37·p36 complex; lane 8, wt p140 and p38, mutant p40·p37·p36 complex (p36K66a); lane 9, wt p140 and p38, mutant p40·p37·p36 complex (p37K84A); lane 10, wt p140 and p38, mutant p40·p37·p36 complex (p40K82A); lane 11, wt p140 and p38, mutant p40·p37·p36 complex (p37K84Ap40K82A). Reactions shown in lanes 12 and 13 were carried out after immunoprecipitation of two levels of the reconstituted mixture (0.5 and 6 μl, respectively) containing wt p140, p38, and wt p40·p37·p36 complex. The reaction shown in lane 14 was carried out in the absence of RFC. Total nucleotide incorporation (pmol) obtained in the above reactions, detected after acid precipitation and liquid scintillation counting, are shown at the bottom of the figure.