Figure 1.

Analysis of rDNA replication by two-dimensional (2D) gel electrophoresis. (A) Genomic structure of the murine rDNA repeat, scaled in bp. The origin of bidirectional replication (OBR) and the replication fork barrier (RFB) flank the 5446-bp ApaL1 restriction fragment analyzed in D (thick line ending with an arrow in the top panel). A filled rectangle marks the hybridization probe used in Figs. 1D and 3A. (B) Replication intermediates detected by 2D gel electrophoresis. Y-shaped DNA junctions at replication forks rise off the diagonal to form the Y-arc. A cone-shaped signal extending from the crest of the Y-arc represents four-way DNA junctions. Positions of unreplicated (UnR) and replicated (R) DNA are marked. (C) First-passage cells were synchronized as described, and released into S phase for 6 h (-HU sample, ie., no HU treatment). HU was then added to 5 mM concentration and samples taken at 3 h (+HU 3h) and 6 h (+HU 6h) during exposure. Nuclei were stained for replication foci (BrdU, 30′ pulse, green), and for DNA (DAPI, blue). Apoptotic changes marked by Annexin V staining do not occur during HU treatment (data not shown). (D) Replication intermediates from replication of the rDNA genes in S phase-synchronized cells, before and during HU treatment for up to 6 h. Slight distortion of the Y-arc in the first panel (+/+, -HU) is due to a tear in the gel. (E) Relative intensities of the Y-arc signal in the different 2D gels (expressed as a densitometric ratio over signal on the gel diagonal to normalize for DNA loading) were plotted as a percentage of the value in S-phase cells before HU treatment. Standard deviations from the mean are too small to be visible.