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Figure 7 | BMC Molecular Biology

Figure 7

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

Figure 7

Origins that do not replicate in HU may normally replicate in late S phase. (A) 2D gel analysis revealed that a Sca I restriction fragment containing AT1045 near its center replicated only slightly in HU, producing a faint Y arc (white arrow; 0 minutes), but replicated more extensively after HU was removed (15–60 minutes), with a weak bubble arc indicative of origin activity evident at 30 and 45 minutes (dark arrows). The diagram underneath the 2D gel pictures illustrates a 6-kb segment of genomic DNA containing the studied Sca I restriction fragment. The scale indicates the nucleotide position along chromosome 1. Genes, along with their names and directions of transcription, are indicated below the scale. The magenta stretch in the middle of the Sca I restriction fragment indicates the central third of that fragment, within which an origin – if present – is likely to be capable of generating detectable bubble arcs [58]. (B) Microarray results for the two probes flanking AT1045. The wild-type (green) signal for the right-hand probe exceeded the threshold of 1.1 at 4 hours, leading to the classification of "very weak". (C) A restriction fragment containing AT2067 was previously tested and shown to have origin activity [20]. However, our microarray results (all signals below 1.0) suggest that AT2067 does not replicate in HU. AT2067 may replicate in late S phase (only after HU removal), as in the case of AT1045. (D) Microarray measurements for the two probes flanking Ori6. DD Dubey, VK Srivastava, AS Pratihar and MP Yadava (submitted for publication; personal communication) used 2D gel electrophoresis to carefully study a 75-kb stretch of chromosome 2. They found six origins (Ori1-Ori6) capable of generating bubble arcs (summarized in Additional File 10). In HU synchronization experiments, they found that Ori6 replicated only after release from HU. This is consistent with our microarray measurements, which show little or no replication of this region in the continued presence of HU. In panels (B)-(D) the small symbols indicating the origins detected experimentally by other laboratories [34] [14] [15] are not shown.

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