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Figure 9 | BMC Molecular Biology

Figure 9

From: Checkpoint independence of most DNA replication origins in fission yeast

Figure 9

Checkpoint-mutation-dependent late replication of subtelomeric sequences but not ribosomal DNA in HU-treated fission yeast cells. The same strains (JLP1164, JLP1257 and JLP1260) were employed as for the microarray experiments. All three strains contain the cdc25-22 mutation, which allowed them to be blocked in G2 by incubation at 36.5°C. The cells were released from the G2 block and further incubated at 25°C for two or four hours in the presence of 15 mM HU. DNA was isolated at the indicated times, digested with the indicated restriction enzymes, and processed for 2D gel electrophoresis. The diagrams under the 2D gel panels show 6-kb stretches containing the studied restriction fragments (as in Fig. 7A). The horizontal axes of the graphs were adjusted to correspond in scale to the restriction fragment diagrams. In this figure there are no colored symbols representing the locations of origins discovered in other laboratories [34] [14] [15], because none of the other laboratories employed microarray probes in these regions. (A) The probe detected a restriction fragment centered on nucleotide position ~4.524 Mb, in the heterochromatic region near the right end of chromosome 2 (Fig. 8B, Additional File 12). Strong Y arcs, and a smear of replication intermediates with altered structures, are evident only in the checkpoint-mutant strains at the 4-hour time point. (B) The probe detected a restriction fragment centered on ars3001 in the rDNA repeats. Bubble arcs are evident only in the samples from wild-type cells, suggesting that the replication checkpoint is required to prevent loss of bubbles under these experimental conditions.

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