Figure 1.

Characterization of the spatial patterns of replication in mouse C127 Cells. (A) Asynchronous cultures were pulse-labeled with BrdU and stained with anti-BrdU antibodies. Shown are deconvolution images of each labeling pattern (I–VI). DAPI shows intense staining at the positions of mouse chromocenters, as well as visible staining at the nuclear periphery. Chromocenters are generally found to associate with nucleoli, visualized as DNA-free areas that are devoid of DAPI or BrdU staining. (B) Asynchronous cultures were pulse-labeled with CldU and chased for the indicated times before they were pulse-labeled with IdU. Sites of CldU (red) and IdU (green) incorporation were visualized using CldU- and IdU-specific antibodies. Representative confocal images from this “pulse-chase-pulse” experiment are shown. Similar to other mammalian cell lines (O'Keefe et al., 1992; Dimitrova and Gilbert, 1999; Dimitrova and Berezney, 2002), DNA synthesis begins at many small, discrete foci in the internal, euchromatic region of the nucleus, excluding the nucleoli (and associated chromocenters) and nuclear periphery region (pattern I). In pattern II, replication continues throughout the euchromatic region, but also is observed in the perinucleolar and nuclear periphery regions. Pattern III is characterized by decreased euchromatic foci in the interior and increased replication foci at the nuclear (and nucleolar) periphery. By the middle of S phase, most euchromatic foci have finished replication and DNA synthesis begins within the pericentric heterochromatin (pattern IV). Thereafter, replication at the nuclear periphery becomes more predominant, coinciding with the replication of a few internal but non-pericentric domains (pattern V). Finally, small numbers of large speckles are observed in both the interior and periphery of the nuclei (Pattern VI). In B, examples of nuclei chased for the indicated time periods are shown. Because each replicon cluster takes 45–60 min to complete DNA synthesis (Ma et al., 1998; Dimitrova and Gilbert, 1999; Leonhardt et al., 2000), there is substantial colocalization of IdU and CldU after 15 min, much less colocalization at 45 min, and no detectable colocalization after 1 h. After longer pulses, some nuclei can be seen to exit S phase into G2 phase and no longer incorporate IdU (e.g., right-most panel; 3 Hrs.). (C) The percentage of each type of CldU-labeled nuclei (I–VII) that had proceeded to subsequent stages of S phase (or to G2 phase) during the chase was scored. Results shown were derived from an experiment in which more than 200 S phase nuclei were scored for each chase period. (D) Schematic diagram approximating the duration of each replication pattern, derived by doubling the time for 50% of nuclei to move from one pattern to the subsequent pattern and taking into account the total length of the C127 S phase (12 h).