FIG. 1.

UV cross-linking of Rpa2 with nascent SV40 DNA. Proteins were photolabeled with nascent DNA within replicating SV40 chromosomes pulse-labeled by BrdUTP and [α-³²P]dATP for 90 s. Following DNase I digestion, Rpa2 was immunopurified and detected by immunoblotting and autoradiography, as described in Materials and Methods. (A) Immunoblot probed with anti-SSB34A. Proteins extracted from a nonlabeled control nuclear monolayer (lane 1), antibody alone (lane 2), the immunoprecipitates of cross-linked proteins derived from the standard reaction mixture (lane 3), a nonirradiated control (lane 4), or a control with dTTP instead of BrdUTP (lane 5) are shown. (B) Autoradiogram of proteins immunoprecipitated from the standard mixture (lane 1), a nonirradiated control (lane 2), or the control with dTTP instead of BrdUTP (lane 3). (C) Immunoprecipitation of the photolabeled RPA heterotrimer. RPA was immunoprecipitated under native conditions from the photolabeled protein mixture derived from replicating SV40 chromatin with anti-SSB34. After separation by SDS-PAGE, individual RPA subunits were detected by immunoblotting and photolabeled derivatives were detected by autoradiography. (D) Densitometric tracings of photolabeled RPA precipitated under native conditions by the indicated MAbs and resolved by SDS-PAGE as described for panel C. H and L indicate heavy and light Ig chains, respectively. The arrows and arrowheads point to the ECL signal of Rpa2 and the photolabeled derivative, respectively. Ctrl, control; Ipc, immunoprecipitated cleared Rpa.