Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE

<div><p>SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, <i>nse3</i>-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). <i>SIR4</i> recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, <i>nse3</i>-1/ <i>sir4</i> Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.</p></div

The <i>nse3</i>-1 allele exhibits genetic interactions with the loss of <i>SIR4</i> and <i>RIF2</i>.

<p>(A) TPE was determined in strains with the <i>URA3</i> reporter at the <i>adh4</i> locus of Chromosome VIIL as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.g003" target="_blank">Fig 3E</a>. Overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates at 34°C in wild type (JC1991), <i>sir4</i>Δ (JC3818), <i>nse3</i>-1(JC3860), <i>nse3</i>-1 <i>sir4</i>Δ (JC3870) isogenic strains. (B) Transcription levels in wild type (JC470), <i>nse3</i>-1 (JC3607), <i>sir4</i>Δ (JC3737), and <i>nse3</i>-1 <i>sir4</i>Δ (JC3741), and (C) <i>rif2</i>Δ (JC2992) and <i>nse3</i>-1 <i>rif2</i>Δ (JC3269) at sub-telomeric genes <i>CHA1</i> and <i>VAC17</i> on Tel3L and <i>YR043C</i> on Tel9R as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref062" target="_blank">62</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref063" target="_blank">63</a>]. Expression values are mRNA levels relative to <i>ACT1</i> and normalization to wild type cells. Error bars represent ± SD of n = 3 experiments with p values < .05 from a two-tailed <i>t</i>-test indicated by (*). (D) Chromatin immunoprecipitation (ChIP) was performed on Rif2^Myc and showed similar levels of recovery in wild type (JC2380) and <i>nse3</i>-1 (JC3235) mutants. (E) ChIP on Smc6^FLAG in wild type (JC1594), <i>rif1</i>Δ (JC2754) and <i>rif2</i>Δ (JC3074) cells with enrichment levels for untagged strains in wild type and mutants shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.s008" target="_blank">S5C and S5D Fig</a>. The mean ± SD of the fold enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) relative to the control (ctrl) late replicating region on Chromosome V (469104–469177) is reported. In <i>rif2Δ</i> mutants the p values < .05 = 0.53 (Tel1L), 0.13 (Tel6R), and 0.15 (Tel15L) indicated that the difference was not significant from wild type. (F) Telomere length was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref015" target="_blank">15</a>]. Southern blot analysis was performed on 1μg XhoI-digested genomic DNA hybridized with a radiolabeled poly (GT/CA) probe in wild type (JC470), <i>nse3</i>-1 (JC3607), <i>rif1</i>Δ (JC3448), <i>nse3</i>-1 <i>rif1</i>Δ (JC3623), <i>rif2</i>Δ (JC2992), <i>nse3</i>-1 <i>rif2</i>Δ (JC3269), <i>rad52</i>Δ (JC1427), <i>nse3</i>-1 <i>rad52</i>Δ (JC3629), <i>rif2</i>Δ <i>rad52</i>Δ (JC3603), and <i>nse3</i>-1 <i>rad52</i>Δ <i>rif2</i>Δ (JC3627) strains.</p

Smc5/6 is a telomere binding complex.

<p>(A) A schematic representation of the Smc5/6 complex showing the location of Nse3 as part of a trimeric sub-complex located at the head region where Smc5 and Smc6 meet. (B) Chromatin immunoprecipitation (ChIP) followed by qPCR was performed on Smc6^FLAG (JC1594) at the indicated time points after release from α-factor. The fold enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) compared to a control (ctrl) late replicating region on Chromosome V (469104–469177) is reported with the mean ± SD for n≥3 experiments performed in technical duplicate. (*) Indicates a statistically significant level of enrichment compared to the ctrl with p values < .05 by a two-tailed <i>t</i>-test. Smc6^FLAG enrichment at Tel1L is higher at 0 and 15 minutes after release, but with p values = 0.08 and p = 0.06 respectively. The lower panels show flow cytometry on ChIP samples with an asynchronous culture shown in black at the 0 time point. (C) Drop assay of exponentially growing wild type (JC470) and <i>nse3</i>-1 (JC3607) cells that were grown for 48 hours at the indicated temperatures on YPAD and 1:5 serial dilutions. (D) Schematic diagram of Nse3. “MHD” represents <u>M</u>elanoma <u>H</u>omology <u>D</u>omain in Nse3 protein. Seven amino acid substitutions in Nse3-1 are shown in red. (E) Chromatin immunoprecipitation (ChIP) on Smc6^FLAG in wild type (JC1594), <i>nse3</i>-1 (JC2630), <i>mms21</i>-11 (JC2075) and the non-tagged (nt) control strains for wild type (JC470), <i>nse3</i>-1 (JC3607), and <i>mms21</i>-11 (JC1879) in asynchronous cultures. The fold enrichment levels are relative to the late-replicating control region on Chr V for n = 3 experiments with the mean ± SD. All primers are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.s002" target="_blank">S2 Table</a>. Enrichment levels for wild type and mutant cells with p values < .05 from a two-tailed <i>t</i>-test are indicated by (*). (F) Telomere length was determined as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref015" target="_blank">15</a>]. Southern blot analysis was performed on 1μg XhoI-digested genomic DNA hybridized with a radiolabeled poly (GT/CA) probe in wild type (JC471), <i>nse3</i>-1 (JC3032), <i>mms21</i>-11 (JC1981), and <i>smc6</i>-9 (JC1358).In higher eukaryotes, telomeres are challenged by the continuous loss of DNA due to the end replication problem. However, in <i>Saccharomyces cerevisiae</i>, telomere length is maintained by the continued expression of telomerase, an enzyme containing a RNA subunit that serves as a template for <i>de novo</i> telomere synthesis [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref016" target="_blank">16</a>]. After the 3’ end is extended by telomerase, the replicative DNA polymerase fills in the complementary strand. Both telomerase extension and semiconservative replication at telomeres are included in the final events of S phase (for review see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref017" target="_blank">17</a>]). In the absence of telomerase activity, telomeres shorten extensively, leading to senescence, however a small percentage of cells survive by extending their telomeres through the HR dependent alternative lengthening of telomeres (ALT) pathway [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref018" target="_blank">18</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.ref021" target="_blank">21</a>].</p

Smc5/6 physically associate with Sir4 and is important for TPE.

<p>(A) Co-immunoprecipitation (Co-IP) as described in the materials and methods section was performed in cells carrying Sir4^Myc and Nse3^HA (JC3736) with Nse3^HA (JC2823) as control or (B) Sir4^Myc and Smc6^FLAG (JC3853) with Smc6^FLAG (JC1594) as a control. (C) ChIP was performed on Smc5^FLAG in wild type (JC3728) and <i>sir4</i>Δ (JC3720) and (D) Smc6^FLAG in wild type (JC1594) and <i>sir4</i>Δ (JC3732) and non-tagged (nt) strains in wild type (JC470) and <i>sir4</i> Δ (JC3737) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006268#pgen.1006268.g001" target="_blank">Fig 1E</a>. The fold enrichment levels are relative to the late-replicating control region on Chr V for n≥3 experiments with the mean ± SD at three native subtelomeres (Tel1L, Tel6R and Tel15L) with p values < .05 from a two-tailed <i>t</i>-test indicated. (E) TPE was determined in strains with the <i>URA3</i> reporter at the <i>adh4</i> locus of Chromosome VIIL. Tenfold (1:10) serial dilutions of overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates at 25°C and 34°C in wild type (JC1991), <i>sir4</i>Δ (JC3818), <i>nse3</i>-1 (JC3860), <i>mms21</i>-11 (JC1080) and <i>smc6</i>-9 (JC1077) isogenic strains.</p