Figure 6.

Uncoupling of MCM helicase and DNA polymerase activities is not sufficient for checkpoint activation. (A) High concentrations of aphidicolin prevent Chk1 phosphorylation. Plasmid DNA was replicated as described in Figure 1A. Aphidicolin was added to achieve a final concentration as shown. Samples were analyzed for phospho-Chk1 (S344) or total Chk1 by immunoblotting at the indicated times. Replication was analyzed in parallel by incorporation of [α³²P]-dCTP into plasmid DNA followed by analysis on chloroquine agarose gels and autoradiography. (B) Addition of a high concentration of aphidicolin in S phase induces immediate Chk1 phosphorylation. The experiment was performed as described in A using plasmid DNA except that aphidicolin was added to a concentration of 1.5 mM to cytosol prior to NPE addition or 25 min post-NPE addition. (C) Monoclonal antibody SJK-132 can both activate and block Chk1 phosphorylation. A stock solution of SJK-132 (22 mg/mL stock) was added to crude interphase extract at 0.2%, 0.8%, or 4% v/v prior to chromatin addition. Sperm chromatin was added to a final concentration of 2000 nuclei/μL. Aphidicolin was added to a final concentration of 100 μg/mL. Samples were taken at the times shown and immunoblotted with antibodies to phospho-Chk1 (S344) and RPA70.