Figure 1.

Replication is slowed in response to UV treatment. (A) Sperm nuclei (2000/μL) were mock-treated or UV-treated and added to Xenopus interphase extract in the presence (+caffeine) or absence (+buffer) of caffeine (4 mM), and the extract was divided into two samples. To assay replication, aliquots were removed from one sample at the given times, incubated with [α-³²P]dCTP for 15 min, terminated, separated on a 0.8% agarose gel, and analyzed by autoradiography. To assay phosphorylation of xChk1, an in vitro translated, [³⁵S]methionine-labeled fragment of xChk1 (Chk1ΔKD) was added to the second sample (5% reaction volume). Nuclei were isolated from this sample at 100 min, then proteins were separated by SDS-PAGE and analyzed by autoradiography. (B) CSF extract (10 μL) was preincubated with recombinant geminin or an equal volume of buffer. The extract was then supplemented with [α-³²P]dCTP and mock- or UV-treated sperm nuclei, incubated at room temperature for 100 min, and processed as described in A.