Figure 4. S phase progression correlates with the sequential activation of replicon clusters as defined by their genetic continuity along individual chromosome.

Cells were pulse-labeled with biotin-dUTP and BrdU separated by 1 h without label (A) and double-labeled DNA fibers of >0.8 Mbp in length collected (B). Typical examples (B) show two major classes, where the 1^st and 2^nd pulse labels were incorporated into genetically adjacent replicon clusters. (B) panels 1–2 show a single fiber that extends over two adjacent imaging fields; the up pointing arrows show part of the replicon cluster labeled with biotin-dUTP during the 1^st pulse; down pointing arrows show BrdU incorporation between two growing replication forks. Panel 3 shows a typical cluster with four active replicons, which were labeled during the 1^st pulse, and two adjacent replicon clusters (defined my multiple Br-labeled tracks) activated during the 2^nd pulse. In other clusters the labeling was confined within a single active cluster that was labeled during both periods of incorporation (Figure S9). To analyze genetic continuity, BrdU incorporation was monitored in the vicinity of stretches of biotin labeled DNA of >0.8 Mbp DNA with labeling properties expected for early S phase replicon clusters (B; n = 50). Double labeled fibers were scored in two classes (C,D): 1) Extending replicons - contained biotin-labeled replicons with internal forks labeled with BrdU during the 2^nd pulse. 2) Clusters with secondary activation - contained multiple BrdU patches in the DNA fiber adjacent to the biotin-labeled cluster. In the same spread fields, fibers containing tracks labeled uniquely with BrdU (ie >250kbp from biotin-labeled tracks; D) were also recorded (C). The sizes of scale bars are shown on individual panels.