Figure 1. Double-labeled replication foci are segregated in specific regions of mitotic chromosomes.

Different dUTP analogues were incorporated into newly replicated DNA and individual chromosomes resolved by random mitotic segregation over 6–7 days (A). Different models (B) show possible relationships between individual DNA foci that are replicated at different times of S phase. In each panel, the replication foci of a single CT (spheres with black rims) and parts of three adjacent CTs (spheres with grey rims) are shown. Foci within the central CT are genetically linked along the chromosome fiber (black zig-zag line). During pulse labeling, some foci are labeled during the 1^st pulse (green) and others during the 2^nd (red). At this time, the alternative models are indistinguishable, with all green foci lying adjacent to neighboring red foci. 6–7 days later, the foci of individual CTs can be visualized as the surrounding CTs are no longer labeled. The innate plasticity of CTs (2 inter-changeable forms are shown) supports distinct predictions about S phase progression: i) if progression is based on spatial continuity of foci at the time of labeling subsequent changes in CT structure will degrade the side-by-side relationship of foci whereas ii) if progression is based on genetic continuity the side-by-side relationship will be preserved. HeLa cells (C) were labeled with AF448-dUTP (green) and Cy3-dUTP (red), grown for 6 days and DAPI-stained chromosome spreads prepared. Deconvolution microscopy shows that 100% (n = 65 chromosomes from 25 metaphase plates) of the labeled chromosomes incorporated both dUTP analogues and that all labeled regions (note that labeling appears in chromosomal bands at this level of resolution) contained both analogues. A merge of the individual channels and a high-resolution merge of the highlighted region (rectangle) are shown to emphasise co-association of the 1^st and 2^nd labels. Diploid human fibroblasts (D) were labeled with biotin-dUTP and BrdU with an intervening unlabeled period of 1h. Labeled chromosomes were resolved by random mitotic segregation (6 days) and confocal imaging performed following indirect immuno-fluorescence using specific antibodies to biotin (red) and BrdU (green). Individual red and green channels and a channel merge were overlaid on the DAPI-stained chromosomes as shown. Merged images with the DAPI removed (D, bottom right panel) were used to demonstrate the co-association of foci along individual chromosomes - the white line highlights the labeled foci along one chromatid of a single chromosome. Scale bars: 10 µm in (C) and 5 µm in (D).