FIGURE 3.

Chk1 phosphorylation in extracts is correlated with the total amount of ongoing replication. A, extracts were prepared across a range of nuclei concentrations and analyzed for Chk1 phosphorylation after 30 min. The asterisk marks an unknown nonspecific band. B, extracts were pretreated with varying amounts of recombinant geminin, and sperm chromatin was added to 10,000 nuclei/μl. Replication per nucleus was determined as in Fig. 1A. C, samples were prepared in parallel to those in B using the same batch of extract, and Chk1 phosphorylation was analyzed. D, extracts were pretreated with geminin at a range of concentrations, as in B, and then supplemented with 2,000 nuclei/μl. DNA synthesis was then measured. A control showing replication at 10,000 nuclei/μl was included for comparison. E, in a parallel experiment to D, Chk1 phosphorylation was analyzed in extracts pretreated with geminin and supplemented with 2,000 nuclei/μl. F, Chk1 activation in the presence of geminin. Extracts were pretreated with geminin and supplemented with 2,000 or 10,000 nuclei/μl. Chk1 phosphorylation was induced in geminin-treated extracts using 30 μm aphidicolin as indicated.