FIG. 4.

Replication fork rates in permeabilized wild-type (WT) and Chk1^−/− DT40 cells in vitro. (A) Wild-type and Chk1^−/− cells were encapsulated in agarose microbeads and permeabilized, and aliquots were then pulse-labeled for 30 min with digoxigenin-11-dUTP. Agarose beads were stained with DAPI to identify nuclei and with fluorescein isothiocyanate (FITC)-tagged antidigoxigenin (anti-Dig) antibody to identify S-phase cells. The fraction of S-phase cells in each population was calculated from multiple microscopic fields and used to normalize replication fork rates, as shown in panel B. The fractions of cells in S phase were 43% (±3%) and 24% (±4%) for wild-type and Chk1^−/− cells, respectively. Note that a single agarose bead is present in the top panels and two are present in the bottom panels. (B) Replication fork rates were quantified in permeabilized wild-type and Chk1^−/− cells encapsulated in agarose microbeads, as described in Materials and Methods, in the presence of [³²P]TTP. Results are the means of three independent experiments, with error bars representing 1 standard deviation.