FIG. 3.

Pulse-chase kinetics of Rpa2’s photolabel. SV40 DNA pulse-labeled for 90 s with BrdUTP and [α-³²P]dATP was chased with dTTP and nonlabeled dATP as indicated. After UV cross-linking, Rpa2 was immunopurified and detected as described in Materials and Methods and in the legend of Fig. 1. (A) Autoradiogram; (B) immunoblot; (C) patterns of nascent SV40 DNA during chase; (D) Rpa2 CI (open squares) and relative labeling (R.L.) of the RDP fraction (filled triangles) versus chase time. The photolabeled protein samples shown in panel A were isolated from the bulk (90%) of the reaction mixture, and the blot showing them was exposed for a week. The total nascent DNA shown in panel C represents 10% of the reaction mixture, and the gel containing it was exposed for 16 h. H and L indicate heavy and light Ig chains, respectively. The arrow points to the ECL signal of Rpa2; the arrowhead points to the photolabeled derivative. M, DNA size markers (in nucleotides).