Identification of a functional domain in Scc3.

<p><b>A.</b> Schematic of the cohesin complex. The two yellow balls between the Smc1 and Smc3 represent ATP molecules. <b>B.</b> Flowchart of the experimental design to identify <i>SCC3</i> dominant negative mutants. <b>C.</b> Strain VG3135 (<i>scc3-6</i>) cells carrying pRS406 (<i>pGALURA3</i>), pIO88 (<i>pGAL</i>-<i>SCC3 URA3</i>) or pIO88R1 (<i>pGAL</i>-<i>scc3-I358insURA3</i>) were grown to saturation in SC-URA media. Fivefold serial dilutions of each strain was plated on SC-URA plates containing either glycerol or galactose and grown at 23°C. <b>D.</b> Sequence alignment of Scc3 was generated by ClustalX. The arrow indicates the insertion site after I358. The blue and red frames indicate the RID A region G350-I359 and the conserved sequence F367-D373, respectively.</p

Analysis of sister chromatid cohesion upon scc3-I358ins overexpression.

<p><b>A.</b> Flowchart of the experimental design. <b>B.</b> Strain YIO81 with plasmid pRS406 (<i>pGAL URA3</i>), pIO88 (<i>pGAL</i>-<i>SCC3 URA3</i>) or pIO88R1 (<i>pGAL</i>-<i>scc3</i>-<i>I358ins URA3</i>) were grown in YEP lactic acid (2%) to mid-log phase and arrested in G1 using α-factor. Galactose was added to a final concentration of 2% for 1 h; cells were then released into the cell cycle and rearrested in G2/M by nocodazole. Sister chromatid cohesion was analyzed by using the GFP spot assay (n = 3). <b>C.</b> Strains analyzed in B were processed for chromatin spreads after cells were arrested in G2/M, as described in the materials and methods section. Cohesin was detected by indirect immunofluorescence with an anti-Mcd1 antibody (n = 2).</p

Analysis of chromosome condensation in Scc3 point mutants.

<p><b>A.</b> Strains YOG3021 (<i>SCC3-6HA scc3-6</i>), YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>) and YOG3025 (<i>scc3-K372A-6HA scc3-6</i>), were grown at permissive temperature (23°C) and arrested at G1 using α-factor. The cells were shifted to restrictive temperature (35.5°C) and re-arrested in the G2/M phase using nocodazole. Nuclei were spread and the bulk DNA was stained with DAPI. The morphology of the <i>rDNA</i>-loop of 100 nuclei of each strain was scored (n = 3). <b>B.</b> Representative photomicrographs of wild-type <i>rDNA</i> loops (left panel) and condensation defects at the <i>rDNA</i> locus. Arrow indicates the rDNA region.</p

Scc3 does not affect the Smc3-Mcd1 interaction.

<p><b>A.</b> Strain YOG3049 (<i>SCC3-AID-V5 SMC3-6HA</i>) was grown to the mid-log phase. Cells were divided into two flasks and 1 mM IAA was added to one of them for 60 minutes. The depletion of Scc3-3V5 was measured in the extract by Western blot with antibodies against V5. Un-depleted and depleted Scc3 cells were used for immunoprecipitation of Smc3-6HA with anti-HA antibodies. The co-precipitation of Mcd1 was detected by anti-Mcd1 antibody. <b>B</b>. Strain YOG3027 (<i>SCC3-AID-V5 SCC2-12MYC</i>) was grown to the mid-log phase. Cells were divided into two flasks and 1 mM IAA was added to one of them for 120 minutes. The depletion of Scc3-3V5 was measured in the extract by Western blot with antibodies against V5. Cells were used for immunoprecipitation of Scc2-12Myc with anti-MYC antibodies. The co-precipitation of Mcd1 was detected by anti-Mcd1 antibody.</p

scc3 Y371 binding to the <i>rDNA</i> is reduced.

<p>Strains YOG3021 (<i>SCC3-6HA scc3-6</i>) and YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: <b>A.</b> chromosome III CARC1; <b>B.</b> chromosome IV centromere; <b>C.</b> chromosoXII <i>rDNA</i>. A representative PCR is shown (n = 3).</p

Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.

<p><b>A.</b> Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (<i>SCC2-3V5</i>), YIO92 (<i>SCC2-3V5 SCC3-6HA</i>) or YIO92R1 (<i>SCC2-3V5 scc3-I358ins-6HA</i>) cells were grown to mid-log phase in YPD media, lysed and subjected to immunoprecipitation against the HA tag (Scc3). Precipitated proteins were analyzed by Western blot using antibodies against HA (IP), Mcd1 (co-IP), V5 (co-IP) and Pds5 (co-IP). <b>B.</b> Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (<i>WPL1-3V5</i>) YOG3007 (<i>WPL1-3V5 SCC3-6HA</i>) or YOG3008 (<i>WPL1-3V5 scc3-I358ins-6HA</i>) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).</p

A Conserved Domain in the Scc3 Subunit of Cohesin Mediates the Interaction with Both Mcd1 and the Cohesin Loader Complex

<div><p>The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized <i>SCC3</i> to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.</p></div

Impact of cohesin accessory factors on Scc2 binding to cohesin.

<p><b>A.</b> Strains YOG3040 (<i>wpl1</i>Δ <i>SCC2-3V5</i>), YOG3002 (<i>wpl1</i>Δ <i>SCC2-3V5 SCC3-6HA</i>) or YOG3003 (<i>wpl1</i>Δ <i>SCC2-3V5 scc3-I358ins-6HA</i>) were grown to the mid-log phase in YEPD media. Cells were lysed and subjected to immunoprecipitation against the HA tag of Scc3. The co-precipitation of Scc2 was detected by anti V5 antibody. <b>B.</b> Strain YOG3050 (<i>PDS5-3V5-AID SCC2-12MYC</i>) YOG3044 (<i>PDS5-3V5-AID SCC2-12MYC SCC3-6HA</i>) and YOG3045 (<i>PDS5-3V5-AID SCC2-12MYC scc3-6HAI358ins-6HA</i>) were grown to the mid-log phase. Cells were divided into two flasks and 1 mM IAA was added to one of them for 60 minutes. The depletion of Pds5-3V5 was measured in the extract by Western blot with antibodies against V5. Cells were used for immunoprecipitation of Scc3 with anti-HA antibodies. The co-precipitation of Scc2 was detected by anti MYC antibody.</p

Cohesion and chromosome binding of scc3-I358Ins under native expression levels.

<p><b>A.</b> Strains YIO81 (<i>scc3-6</i>), YIO91 (<i>SCC3-6HA scc3-6</i>) andYIO91R1 (<i>scc3-I358ins-6HA scc3-6</i>) were grown to saturation in YPD media. Fivefold serial dilutions of each strain was plated on YEPD plates and grown at either the permissive (23°C) or restrictive (37°C) temperature for <i>scc3-6</i>. <b>B</b>. Flowchart of the experimental design to score sister chromatid cohesion. <b>C.</b> Strains as indicated in A were grown at 23°C to mid-log phase and arrested in G1 using α-factor. Cells were then shifted to 35.5°C for 1 h, released into the cell cycle and re-arrested in G2/M with nocodazole. Samples for the cohesion assay were taken approximately every 15 minutes (n = 3). The frame indicates S-phase of the cell cycle as determined by flow cytometry (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005036#pgen.1005036.s001" target="_blank">S1 Fig</a>). <b>D.</b> Strains described in A were processed as in B for chromatin immunoprecipitation analysis. Scc3, HA tagged proteins were immunoprecipitated. Precipitated DNA was analyzed by quantitative PCR with six primer pairs for the MAT CAR and CARC1, as described (Material and methods). A representative experiment is shown (n = 3). <b>E.</b> Strains described in A were processed as in B for chromatin immunoprecipitation analysis. Mcd1 was immunoprecipitated. Precipitated DNA was analyzed by quantitative PCR for CARC1. A representative experiment is shown (n = 3).</p

Analysis of alanine point mutations in Scc3’s SCD.

<p><b>A.</b> Strain YIO81 (<i>scc3-6</i>), carrying an ectopic copy of <i>SCC3-6HA</i> (YOG3021), <i>scc3-F367A-6HA</i> (YOG3022), <i>scc3-R370A-6HA</i> (YOG3023), <i>scc3-</i>Y371A-6HA (YOG3024), <i>scc3-K372A-6HA</i> (YOG3025) or <i>scc3-D373A</i>-<i>6HA</i> (YOG3026) were grown to saturation in YPD media. Tenfold serial dilution of the strains were plated on YPD plates and grown at either permissive (23°C) or restrictive (35°C) temperature. <b>B</b>. Strains YIO81 (<i>scc3-6</i>), YOG3021 (<i>SCC3-6HA scc3-6</i>), YOG3024 (<i>scc3-Y371A-6HA scc3-6</i>), YOG3025 (<i>scc3-K372A-6HA scc3-6</i>) and YOG3026 (<i>scc3-D373A-6HA scc3-6</i>) were analyzed for sister chromatid cohesion using the GFP spot assay (n = 3). Cell cycle progression was determined by flow cytometry analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005036#pgen.1005036.s004" target="_blank">S4 Fig</a>). <b>C</b>. Co-immunoprecipitation of Mcd1 with Scc3-6HA, scc3-Y371A-6HA, scc3-K372A-6HA or scc3-D373A-6HA. Precipitation of Scc3 was done by using an anti-HA antibody.</p