Chr XII replication defect in Nse5-Smc6 double degron cells is improved by Fob1 removal.

<p>A. Diagram showing the rDNA array on Chr XII and key features of each rDNA repeat. Fob1 binds to the RFB in each rDNA repeat to block replication forks moving opposite the direction of 35S rDNA transcription. Replication of an rDNA repeat begins at the rARS replication origin. Note that XhoI digests an intact rDNA array out of Chr XII. The probe used for detecting Chr XII and rDNA on Southern blots is depicted. B. FACS profiles showing Nse5-Smc6 double degron (<i>FOB1</i>) and Nse5-Smc6 double degron <i>fob1∆</i> (<i>fob1∆</i>) cells progressing through the first cell cycle of a G1-arrest and release assay as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g002" target="_blank">Fig 2A</a>. C. Western blotting with anti-BrdU antibody of PFGE-separated chromosomes shows that <i>fob1∆</i> increases in-gel Chr XII BrdU signals in degron cells progressing through the first cell cycle. D. Southern blots for Chr XII and Chr III showing that the <i>fob1∆</i> effect is Chr XII-specific. Chr XII and Chr III signals in-gel and in wells were detected using radiolabeled probes against rDNA or the ARS305 region of Chr III, respectively. Percentage of chromosome gel entry was calculated as described. Standard deviations and P-values (t-test, *p<0.05) are derived from n = 6 trials for rDNA and n = 3 trials for ARS305. E. <i>fob1∆</i> reduces the rDNA replication defect in Nse5-Smc6 double degron cells. Chromosomes were digested with XhoI before PFGE and then the rDNA array was detected by Southern blotting with an rDNA-specific probe. Percentage of rDNA gel entry was calculated as described in (D). Standard deviations and P-values (t-test, *p<0.05) are derived from n = 3 trials for rDNA.