FIG. 5.

Phosphorylation of pol-prim by cyclin A/cdk2 inhibits initiation and elongation of SV40 DNA replication. (A and B) pol-prim purified after prephosphorylation (Phos) with cyclin A/cdk2 (A/2) or mock phosphorylation (Mock) was analyzed by SDS-PAGE with 10% (A) or 7.5% (B) polyacrylamide and staining with Coomassie blue (A) or Western blotting with a p68-specific antibody (B). The mobilities of marker proteins (M) of known sizes are indicated (in kilodaltons) at the right. (C) Initiation and elongation of SV40 DNA replication was assayed in reaction mixtures containing SV40 origin DNA, topoisomerase I, RPA, T antigen, and increasing amounts (5, 10, 15, and 20 primase units) of mock-phosphorylated pol-prim (lanes 1 to 4) and cyclin A/cdk2-phosphorylated pol-prim (lanes 5 to 8). Reaction products made in the absence of T antigen (lane 9) or pol-prim (lane 10) are shown. Radiolabeled elongation products synthesized by pol-prim were detected by alkaline electrophoresis and autoradiography. The radioactivity (counts per minute [cpm]) incorporated in each reaction is shown below the lane. The mobilities of end-labeled marker DNA fragments of the indicated lengths in nucleotides are shown at the right.