Chr XII replication is affected in the first cell cycle after Smc5/6 depletion.

<p>A. Experimental scheme of alpha factor (aF)-induced G1 arrest followed by release into cycling for examination of chromosomal replication. This procedure was used for subsequent figure panels unless otherwise noted. B. FACS profiles of IAA-treated Nse5-Smc6 double degron and control (TIR alone) cells after G1 arrest and synchronized release into first cell cycle progression. C. Western blot showing reduced BrdU incorporation for Chr XII in degron versus control cells in the first cell cycle. Chromosomes were separated by PFGE and new DNA synthesis was detected using an anti-BrdU antibody. D. FACS profiles of G1 arrest and release assays using Nse5-Smc6 double degron treated or not treated with 1 mM IAA. E. Western blot showing reduced Chr XII BrdU incorporation in IAA-treated cells compared with untreated cells in the first cell cycle after G1 release. New DNA synthesis in each chromosome was detected using an anti-BrdU antibody after PFGE. Samples in D and E were run on the same gel, with dotted lines indicating the junction that separates samples from the two strains. The same labeling convention is used for subsequent figures showing PFGE data.</p

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.</p

<i>fob1Δ mph1Δ</i> improves rDNA replication in Nse5-Smc6 degron cells.

<p>A. FACS profile showing first cell cycle progression of Nse5-Smc6 double degron (<i>FOB1 MPH1</i>) and Nse5-Smc6 double degron <i>fob1Δ mph1Δ</i> (<i>fob1Δ mph1Δ</i>) cells. Experiments followed the scheme shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g002" target="_blank">Fig 2A</a>. B. XhoI-digested samples were subjected to PFGE and Southern blot to examine rDNA array replication. Signals from an rDNA-specific probe are quantified as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g003" target="_blank">Fig 3E</a>. Standard deviations and P-values (t-test, *p<0.05) are derived from n = 2 trials. C. Combined graph of rDNA signals derived from XhoI digested DNA separated by PFGE followed by Southern blotting shows that <i>fob1Δ mph1Δ</i> and <i>fob1Δ</i> have similar levels of suppression of rDNA replication in Nse5-Smc6 degron cells. Quantifications for wild-type and Nse5-Smc6 double degron cells without IAA treatment are included; representative images and FACS analyses are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.s005" target="_blank">S5 Fig</a>.</p

<i>fob1Δ</i> and <i>mph1Δ</i> reduce recombination structures at rDNA in Nse5-Smc6 degron cells.

<p>A. Schematic of DNA structures detected by 2D gel for the indicated BglII fragment of rDNA repeat. Top: 1N and 2N dots indicate linear DNA. Y-arc represents fork. Replication fork arrested on RFB is shown as a dot on Y-arc. X-shape structures (or X-mol) represent recombination structures (arrow). Bottom: the BglII fragment and the probe for Southern blot are shown. B. Cells from the indicated strains were collected at 60 min and 120 min after G1 release, following the scheme shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g002" target="_blank">Fig 2A</a>. Genomic DNA was isolated, digested with BglII, and analyzed on 2D gel followed by Southern blotting with a probe depicted in (A). Arrows indicate the X-mol DNA that show an increase in degron cells compared with control cells. C. Quantification of X-mol structures at 60 min and 120 min after G1 release. Signals in the control cells were standardized to 1, and the fold-differences of other strains with respect to the control are shown. Mean and standard deviation are derived from n = 3 trials. Statistical differences between the values of degron cells and those containing <i>fob1Δ</i>, <i>mph1Δ</i>, and <i>fob1Δmph1Δ</i> were calculated by Student’s t-test (*p<0.05). D. A model of Smc5/6 function at rDNA. Smc5/6, known to be localized to rDNA, can antagonize Mph1-mediated recombination. Without Smc5/6, paused forks can engage in Mph1-mediated fork regression and subsequent recombination. As Smc5/6 is also required for supporting the STR complex in dissolving recombination intermediates, when Smc5/6 is removed, recombination intermediates can accumulate thus impeding replication completion and subsequent DNA segregation.</p

Chr XII replication is affected in the first cell cycle after Smc5/6 depletion.

<p>A. Experimental scheme of alpha factor (aF)-induced G1 arrest followed by release into cycling for examination of chromosomal replication. This procedure was used for subsequent figure panels unless otherwise noted. B. FACS profiles of IAA-treated Nse5-Smc6 double degron and control (TIR alone) cells after G1 arrest and synchronized release into first cell cycle progression. C. Western blot showing reduced BrdU incorporation for Chr XII in degron versus control cells in the first cell cycle. Chromosomes were separated by PFGE and new DNA synthesis was detected using an anti-BrdU antibody. D. FACS profiles of G1 arrest and release assays using Nse5-Smc6 double degron treated or not treated with 1 mM IAA. E. Western blot showing reduced Chr XII BrdU incorporation in IAA-treated cells compared with untreated cells in the first cell cycle after G1 release. New DNA synthesis in each chromosome was detected using an anti-BrdU antibody after PFGE. Samples in D and E were run on the same gel, with dotted lines indicating the junction that separates samples from the two strains. The same labeling convention is used for subsequent figures showing PFGE data.</p

<i>mph1Δ</i> reduces rDNA replication defects in Nse5-Smc6 degron cells.

<p>A. FACS profile showing first cell cycle progression of Nse5-Smc6 double degron (<i>MPH1</i>) and Nse5-Smc6 double degron <i>mph1Δ</i> (<i>mph1Δ</i>) cells using the same strategy shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g002" target="_blank">Fig 2A</a>. B. Southern blots for Chr XII and Chr III showing that <i>mph1Δ</i> improves Chr XII replication. Chr XII and Chr III signals in wells and in-gel were detected, and percentage of chromosome gel entry was calculated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g003" target="_blank">Fig 3D</a>. Standard deviations and P-values (t-test, *p<0.05, ** p<0.01) are derived from n = 3 trials for rDNA and n = 3 trials for ARS305. C. <i>mph1Δ</i> reduces rDNA replication defect in Nse5-Smc6 double degron cells. Chromosomes were digested with XhoI before PFGE and Southern blot as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g003" target="_blank">Fig 3E</a>. Percentage of rDNA gel entry was calculated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007129#pgen.1007129.g003" target="_blank">Fig 3E</a>. Standard deviations and P-values (t-test, *p<0.05) are derived from n = 3 trials.</p

Smc5/6 loss of function is enhanced by combining degrons.

<p>A. Growth assessment of strains containing AID-tagged Smc5/6 subunits with or without TIR. Ten-fold serial dilutions of log phase cells were spotted onto medium with or without IAA. Tagging each of the seven indicated subunits slows cell growth to different degrees only in the presence of IAA and TIR1. B. Examination of protein degradation in degron strains by Western blot. IAA-treated (90 min) and un-treated asynchronous cultures were examined. All AID subunits tested achieved protein loss. C. Combining Nse5 and Smc6 degron alleles results in greater growth defects than the Smc6 degron alone can achieve. The same number of cells for each indicated strain was plated on media containing 1 mM IAA and grown at 30°C for 2 days. The percentages indicate the average number of colonies formed in each strain relative to TIR1-only controls. D. Experimental scheme and cell cycle progression of Nse5-Smc6 double degron cells. For +IAA samples, cells were arrested in G1 phase by alpha factor, treated with 1 mM IAA for 90 min, released from G1 arrest into fresh IAA-containing media and allowed to progress through the first cell cycle under protein depletion conditions as assayed by FACS analyses. The procedure for–IAA samples was the same, except no IAA was added. E. The Nse5-Smc6 double degron strain was examined for protein degradation and markers of cell cycle progression and DNA damage. Western blots were performed on samples harvested from the time course experiments (D). Protein levels in IAA-treated cells are expressed as a relative percentage of those for untreated cells at each time point after G1 release. Molecular markers of cell cycle progression (Clb2) and DNA damage (Rad53 and γH2A) were examined. Note that both Nse5 and Smc6 protein levels are lower in G1 than in S phase for untreated cells.</p

The loss of Rnr1 activity interferes with the maintenance of short telomeres and accelerates replicative senescence and survivor formation in telomerase-negative cells.

<p><b>A)</b> Experimental approach: Following meiotic segregation and tetrad dissection spore colonies were diluted in liquid medium to an OD₆₀₀ value of 0.02. After every 24h of growth at 30°C the optical cell density has been measured and cells were re-diluted to an OD₆₀₀ of 0.02. The number of cell divisions/generations was calculated as log₂ (OD₆₀₀24h/0.02). <b>B)</b> Senescence curves reflecting the cell density reached after 24 hours of growth as a function of cell divisions (generations). In <i>rnr1</i>Δ <i>crt1</i>Δ <i>est2</i>Δ mutants the onset of replicative senescence and survivor formation is accelerated when compared to <i>crt1</i>Δ <i>est2</i>Δ and <i>est2</i>Δ controls. The number of cell divisions the spore colony went through before the first dilution has been estimated as 25 generations (starting point of the curves). Data is shown as mean +/- SEM (n = 3–5). <b>C)</b> Telomere length analysis by Southern blotting of one biological replicate of each genotype shown in <b>(B)</b>. Telomeres of <i>est2</i>Δ and <i>crt1</i>Δ <i>est2</i>Δ controls shortened progressively with ongoing generations (G) and showed a heterogeneous length indicative of survivor formation at 65 and 62 cell divisions, respectively. The telomeres of <i>rnr1</i>Δ <i>crt1</i>Δ <i>est2</i>Δ mutants could not be maintained after the first dilution and showed a heterogeneous length already at generation 40.</p

In the absence of Rnr1 telomeres cannot become elongated by increased telomerase activity.

<p><b>A)</b> Telomere length analysis by Southern blotting shows that mutations of the long <i>TLM</i> genes <i>PIF1</i> or <i>ELG1</i> cause telomere elongation when introduced in a wild type background but do not affect the short telomere length of <i>rnr1</i>Δ <i>crt1</i>Δ mutants. Telomere blots were performed approximately 200 generations after the long <i>tlm</i> mutation has been introduced. Represented are biological replicates of the indicated strains. <b>B)</b> Telomere length analysis by Southern blotting of cells expressing Cdc13-Est1 and Cdc13-Est2. Expression of the fused proteins results in telomere elongation in wild type cells and <i>crt1</i>Δ mutants but not in <i>rnr1</i>Δ <i>crt1</i>Δ mutants or <i>rnr1</i>Δ <i>crt1</i>Δ <i>elg1</i>Δ mutants. A plasmid expressing Cdc13 was used as a control and did not cause elongation of wild type cell telomeres. Represented are biological replicates of the indicated strains approximately 200 generations after transformation of the indicated plasmid.</p

The Mec1^ATR-Dun1 pathway promotes telomere over-elongation via Rnr1 activation through Sml1 degradation.

<p><b>A)</b> dNTP pools and dGTP ratios (in %) were measured in the indicated strains. <i>dun1</i>Δ mutants show low dNTP levels, which can be elevated above wild type levels by a co-deletion of <i>SML1</i>. In <i>sml1</i>Δ <i>elg1</i>Δ <i>dun1</i>Δ mutants dNTP levels are elevated approximately 4 fold relative to wild type. Mean values +/-SEM are indicated. All values are shown as fold change over the individual dNTP levels of the wild type which were taken from the experiment in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007082#pgen.1007082.g001" target="_blank">Fig 1C</a> (set to 1). n(wt) = 9; n(<i>elg1</i>Δ) = 2; n(<i>sml1</i>Δ) = 2; n<i>(sml1</i>Δ <i>elg1</i>Δ) = 2; n(<i>dun1</i>Δ) = 2; n(<i>sml1</i>Δ <i>dun1</i>Δ) = 2; n (<i>dun1</i>Δ <i>elg1</i>Δ) = 2; n(<i>sml1</i>Δ <i>dun1</i>Δ <i>elg1</i>Δ) = 2. <b>B)</b> Telomere length analysis by Southern blotting. A deletion of <i>DUN1</i> results in short telomeres, which only slightly become elongated by a co-deletion of the long <i>TLM</i> gene <i>ELG1</i>. The simultaneous deletion of <i>SML1</i> restores telomere length in <i>dun1</i>Δ mutants and rescues the over-elongation of telomeres in <i>dun1</i>Δ <i>elg1</i>Δ double mutants. Represented are biological replicates of the indicated strains after approximately 200 generations. <b>C)</b> dNTP pools and dGTP ratios (in %) were measured in the indicated strains. <i>dun1</i>Δ mutants show low dNTP levels, which can be elevated above wild type levels by a deletion of <i>CRT1</i> or simultaneous deletions of <i>CRT1</i> and <i>ELG1</i>. Mean values +/-SEM are indicated. All values are shown as fold change over the individual dNTP levels of the wild type which were taken from the experiment in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007082#pgen.1007082.g001" target="_blank">Fig 1C</a> (set to 1). The dNTP levels of <i>elg1</i>Δ and <i>dun1</i>Δ mutants were taken from the experiment in <b>(A)</b> and are shown as a comparison. n(wt) = 9; n(<i>elg1</i>Δ) = 2; n(<i>elg1</i>Δ <i>crt1</i>Δ) = 2; n<i>(crt1</i>Δ) = 2; n(<i>dun1</i>Δ) = 2; n(<i>dun1</i>Δ <i>crt1</i>Δ) = 2; n(<i>dun1</i>Δ <i>elg1</i>Δ) = 2; n(<i>dun1</i>Δ <i>elg1</i>Δ <i>crt1</i>Δ) = 2. <b>D)</b> Telomere length analysis by Southern blotting. A co-deletion of <i>CRT1</i> does not restore telomere length in <i>dun1</i>Δ mutants nor does it rescue the over-elongation of telomeres in <i>dun1</i>Δ <i>elg1</i>Δ double mutants. Represented are biological replicates of the indicated strains after approximately 200 generations.</p