Stepwise Activation of the ATR Signaling Pathway upon Increasing Replication Stress Impacts Fragile Site Integrity
Figure 2
Moderate fork slowing is not associated with formation of ssDNA foci.
(A) ssDNA detection scheme. Cells hemi-substituted throughout their genome after growth in the presence of CldU for about 1.5 cell generations were treated or not with aphidicolin or HU for different periods of time prior to fixation. CldU was immuno-detected without DNA denaturation, which only permits visualization of substituted and single-stranded regions. (B) Co-detection of ssDNA (red) and chromatin bound PCNA (green) by immunostaining of untreated cells (-) and cells treated as indicated. Nuclei were counterstained with DAPI. (C) Typical pattern of PCNA and CldU foci in cells treated with HU 1 mM during 1 h. (D) Typical pattern of PCNA and CldU foci in cells depleted of ATR and treated with 0.3 µM of aphidicolin for 4 h.