Figure 7. ATM is not involved in the replication checkpoint.

Two asynchronous cultures of AT8052 cells were pulse-labelled for 10 min with CldU, aphidicolin (Aph) was added to one of them (a), and both cultures were grown for a further 60 h. At various time points after CldU labelling (0, 6, 12, 24, 36, 48 and 60 h), aliquots of cells from each culture were washed free of drugs, pulse-labelled with IdU, and then fixed and stained with anti-CldU (green) and anti-IdU (red) antibodies as in Fig. 2. In the presence of aphidicolin, cells were arrested in S phase and the IdU label co-localized with the CldU label in nuclei at all stages of S phase (panels 1 and 3 show examples of early-S-phase nuclei; panel 2 shows a late-S-phase nucleus) throughout the entire chase period. As AT8052 is a primary cell line that could potentially enter into senescence, it was important to verify that cells would have continued to cycle during the chase period. In the absence of aphidicolin (b), the control cell culture grew normally. The size of the nuclei and the type of the IdU replication pattern were used as criteria to evaluate the progression of cells through the cell cycle. During cycle I, the CldU and IdU labelling patterns co-localized at 0 h (panel 4), but had become separated by 6 h (panels 5 and 6) and, at 12 h, large, IdU-negative G2 nuclei (panel 7) and mitotic figures (panel 8) were observed. The appearance of small, CldU-labelled, IdU-negative daughter nuclei (panel 9), often positioned adjacent to each other, indicated progression of cells into the G1 phase of cycle II by 24 h. Later, at 24–48 h, double-labelled S-phase nuclei (panels 10–12) and large, CldU-labelled G2 nuclei (panel 13) re-appeared as the cells moved through cycle II. The appearance of small, IdU-negative nuclei with gaps (arrows) in the CldU labelling pattern (panel 14) indicated that, by 48 h, cells had progressed into cycle III. Gaps correspond to the territories of unlabelled chromosomes⁴⁰,⁴¹, which were generated after two cell divisions (the mechanism responsible for generation of unlabelled chromosome territories is schematically outlined in the two lower-right boxes). Nuclei with unlabelled chromosome territories were also found to have entered S phase (panels 15–17) and G2 phase (panel 18) of cycle III by the 60-h time point.