Figure 5.

Locating CAF-1 at late replication foci relative to a defined DNA synthesis event as a function of time. Sites of DNA synthesis in a synchronous population of HeLa cells in late S phase were first labeled in vivo by BrdU pulse (red). Cells were harvested, immediately or after different chase times. CAF-1 p60 subunit was localized using a specific pAb (green). Samples were analyzed by confocal microscopy. A, A digital merge of single optical sections corresponding to both labels is shown for each chase time. B, Magnification of selected foci showing both labels (from windows in A) are shown. C, Stacks of confocal acquisitions from pulse–chase experiments, similar to those presented in A, were collected for series of nuclei and processed as in Fig. 4 C (3D analysis). Each measurement corresponds to the average behavior of at least 100 foci from one nucleus. The plot represents the BrdU signals colocalizing with CAF-1 in arbitrary units [(C) / (C + R), ratio normalized as described in Materials and Methods] as a function of chase time. Error bars correspond to the maximum variance experimentally obtained.