Replication patterns succeed in an ordered sequence throughout S phase. (*A*) Synchronized cells were released into S phase and pulse labelled with BrdU before harvesting at hourly intervals. Thus, time point 1 refers to cells harvested 1 h after hydroxyurea release. For each time point, a minimum of 200 cells were analyzed. The nuclei were sorted by pattern and the results quantified as percentages of the total number of BrdU- labelled cells. The graph demonstrates an ordered succession of replication patterns as cells progress through S phase with each pattern showing a distinct peak. It is also evident that the overlap between the earlier replication patterns (*Patterns 1* and *2*) and the later patterns (*4* and *5*) is minimal, i.e., always <6%. At later time points, no additional clearly defined peaks were observed (not shown). (*B*) We analyzed ∼50 nuclei per replication pattern randomly selected from an asynchronous cell population pulse labelled with BrdU (10 μM final concentration; 30 min). For each nucleus, the BrdU signal was quantified in equatorial confocal sections (refer to *Materials and Methods*). Although the internal compartment corresponds to 71.55% of the inner nuclear volume, in an equatorial section it is represented by the inner 80% of the nuclear profile area. Thus, the expected proportion of the BrdU signal in the internal compartment, if its distribution was homogeneous, would equal 0.8. The graph represents the average proportion between the area occupied by the BrdU signal in the inner 80% of the nuclear profile and the total area of the signal in each group. Using ANOVA followed by Scheffe's multicomparison tests, the only statistical identities occur for patterns 1 and 2 (*P* = 0.999) and for patterns 4 and 5 (*P* = 0.463). *P* values for other pairs are <0.0001. All the measured proportions are statistically different from expected for a homogeneous distribution as assessed by Student's *t* test (*P* < 0.0001). A significant reduction in the BrdU signal in the nuclear interior is seen as cells progress through S phase.

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