JCS01412 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. GFP-PCNA foci appear at sites adjacent to sites of previous DNA synthesis during early S phase, whereas DNA remains stably positioned. The figure shows a nucleus labelled according to the scheme depicted in Fig. 1a at the indicated time points (minutes) after microinjection (red, Cy3; green, GFP; yellow; colocalization). The nucleus displays a labelling pattern typical for early S phase with many foci throughout the nuclear interior. The boxed regions in a are shown enlarged in the lower panels b and c. The single GFP  and Cy3 fluorescence and the corresponding merged images are shown. Although the Cy3 labelling pattern shows no major rearrangements during S-phase progression GFP-PCNA foci appear at adjacent sites. The arrowhead points to a region between two Cy3-labelled foci. Although this region is also depleted in GFP fluorescence at 45 minutes, a GFP-labelled focus appears in this region 70 minutes after microinjection. (c) Single light optical sections of the nucleus at 400 minutes after microinjection showing the Cy3 and GFP fluorescence (nucleus rotated about 100 degrees over to the right). The Cy3-labelled DNA was still arranged into the pattern typical for early S phase at this time point but the GFP pattern reflects the normal progression through S phase. Only a few replicating sites are left and most of the GFP-PCNA is already diffusely distributed. Bar in a, 5 mm; b, 0.5 mm; and c, 10 mm.

• Supplemental Figure 2 -

  Fig. S2. The panels show a mid-nuclear plane of a daughter nucleus labelled according to the scheme depicted in Fig. 4a. The mother cell has been labelled during mid S phase, as indicated by the Cy3 labelling pattern (red). The daughter nucleus has been imaged during a relatively early stage of S phase, as indicated by the GFP-PCNA pattern (green). During this relatively early stage of the second S phase no incorporation of free Cy3-dUTP at GFP-PCNA foci is observed. Bar, 5 mm.

• Supplemental Figure 3 -

  Fig. S3. After synchronization with mimosin at G1/S and release, HeLa S6 nuclei were first microinjected with Cy3-dUTP (red) and 1.5 hours later with Cy5-dUTP (blue). The nucleus shown in the upper row was labelled with BrdU (green) at about 1.5 hours after microinjection of Cy5-dUTP. All three labels were incorporated into typical S-phase patterns and the different patterns indicate normal progression through S phase. The nucleus shown in the second row was labelled with BrdU on the next day during the second S phase. BrdU was also incorporated normally here. These data, together with the finding that BrdU and GFP-PCNA label identical sites (Leonhardt et al., 2000a) demonstrate that GFP-PCNA foci in triple-labelled nuclei reliably represent functional replication foci.

• Supplemental Table 1 -

  Table S1. Cy3- and GFP-PCNA labelling patterns of class I-III daughter nuclei were assigned to five different temporal stages of S phase (I-V) as defined previously (Sadoni et al., 1999). Note that Cy3- and GFP-PCNA foci in individual class I and II nuclei are not necessarily arranged in the same type of pattern, but can also reflect subsequent temporal stages (compare Fig. 9).

• Supplemental Data (Microsoft Word)