Figure 1.

Characterization of checkpoint activation using plasmid DNA. (A) Initiation of replication on plasmid DNA is required for Chk1 phosphorylation in response to aphidicolin and UV damage. Plasmid DNA was left untreated or pretreated with UV irradiation (1000 J/m²), incubated in cytosol for 30 min, and then transferred to an equal volume of NPE in the presence or absence of aphidicolin (13 μM) as shown. Geminin was preincubated in cytosol at a concentration of 2 μM for 15 min prior to DNA addition, and p27^KIP was added to NPE at a concentration of 10 μM immediately before addition to cytosol. Samples were analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting. Replication was analyzed in parallel by incorporation of [α³²P]-dCTP into plasmid DNA followed by agarose gel electrophoresis and autoradiography. (B,C) Immunodepletion of Rad1, ATRIP, or Claspin. NPE was incubated with rabbit IgG (mock), α-Rad1, α-ATRIP, or α-Claspin antibodies, and the levels of each protein in depleted extracts were analyzed by immunoblotting. (D,E) Rad1, ATRIP, and Claspin are required for plasmid-mediated checkpoint activation. Depleted extracts were used to replicate plasmid DNA in the presence or absence of aphidicolin as described in A. Samples were taken post-NPE addition at the indicated times and immunoblotted as in A. (Lanes 9,10) Recombinant Claspin (6 nM) was added to cytosol after immunodepletion.