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 Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair

<div><p>Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 <i>in vivo</i>. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.</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

Effects of <i>mre11</i>, <i>sae2</i>, <i>exo1</i> and <i>rad51</i> on MMS-induced mono- and diubiquitination of PCNA.

<p>Overnight cultures were subcultured and allowed to grow to a cell count of approximately 1×10⁷cells/ml before being treated with 0.05% MMS (as indicated) for 90 minutes. Total cell extracts were obtained under denaturing conditions and analyzed by SDS-PAGE and western blot. Strains used were HK578-10A (wild type) and its isogenic derivatives WXY994 (<i>pol30-K164R</i>), WXY2959 (<i>siz1</i>Δ), WXY2995 (<i>mre11</i>Δ <i>siz1</i>Δ), WXY2962 (<i>sae2</i>Δ <i>siz1</i>Δ), WXY2963 (<i>exo1</i>Δ <i>siz1</i>Δ) and WXY2994 (<i>rad51</i>Δ <i>siz1</i>Δ). Ub₁ refers to monoubiquitinated PCNA. Ub₂ refers to diubiquitinated PCNA.</p

<i>EXO1</i> belongs to the error-free PRR pathway.

<p>(A) <i>mms2</i> is epistatic to <i>exo1</i> but <i>rev3</i> and <i>exo1</i> are additive. (B) <i>sae2</i> is epistatic to <i>exo1</i>. Strains used are BY4741 and its isogenic derivatives. Experimental conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109292#pone-0109292-g001" target="_blank">Figure 1</a>.</p

<i>SAE2</i> belongs to the yeast PRR pathway.

<p>(A,B) <i>mms2</i> and <i>rev3</i> are epistatic to <i>sae2</i> as judged by a serial dilution assay. (A) <i>sae2</i> vs. <i>mms2</i> or <i>rev3</i>. (B) <i>sae2</i> vs. <i>mms2 rev3</i>. Strains used in (A) and (B) are isogenic derivatives of BY4741. (C) Inactivation of <i>SAE2</i> partially rescues <i>rad18</i> sensitivity to DNA damage. Strains used in (C) are HK578-10A (wild type) and its isogenic derivatives WXY2975 (<i>sae2</i>Δ), WXY930 (<i>rad18</i>Δ) and WXY3008 (<i>rad18</i>Δ <i>sae2</i>Δ). Experimental conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109292#pone-0109292-g001" target="_blank">Figure 1</a>.</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

A proposed working model for the budding yeast PRR pathways.

<p>MRX, in conjunction with Sae2, functions upstream of PCNA monoubiquitination by ssDNA resection thus promoting Rad6-Rad18 to monoubiquitinate PCNA. Exo1 processes ssDNA gaps in the 5′-3′ direction, which facilitates PCNA polyubiquitination by Rad5-Ubc13-Mms2 and subsequent error-free lesion bypass mediated by the Shu complex, HR and Sgs1-Top3 resolution.</p

Genetic interactions between <i>mre11</i> and PRR pathway mutations.

<p>(<b>A,B</b>) <i>pol30-164R</i> is epistatic to <i>mre11</i>. (A) A serial dilution assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109292#pone-0109292-g001" target="_blank">Figure 1A</a>. (B) A liquid killing assay. The results are the average of four independent experiments with standard deviations as shown. Yeast strains used: DBY747 (wild type), WXY2379 (<i>mre11</i>Δ), WXY2384 (<i>pol30-K164R</i>) and WXY2389 (<i>pol30-K164R mre11</i>Δ). All strains used are isogenic to DBY747. (C) Genetic interactions between <i>mre11</i> and <i>mms2 rev3</i> by a serial dilution assay. Experimental conditions were as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109292#pone-0109292-g001" target="_blank">Figure 1A</a>. Yeast strains used: BY4741 (wild type), BY4741 <i>mre11</i>Δ, WXY2536 (<i>rev3</i>Δ <i>mms2</i>Δ) and WXY2528 (<i>mre11</i>Δ <i>rev3</i>Δ <i>mms2</i>Δ). All strains used are isogenic to BY4741.</p