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A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex.

Orgil O, Matityahu A, Eng T, Guacci V, Koshland D, Onn I - PLoS Genet. (2015)

Bottom Line: Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader.These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion.These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine in The Galilee, Bar-Ilan University, Safed, Israel.

ABSTRACT
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 SCC3 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.

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Analysis of sister chromatid cohesion upon scc3-I358ins overexpression.A. Flowchart of the experimental design. B. Strain YIO81 with plasmid pRS406 (pGAL URA3), pIO88 (pGAL-SCC3 URA3) or pIO88R1 (pGAL-scc3-I358ins URA3) 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). C. 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).
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pgen.1005036.g002: Analysis of sister chromatid cohesion upon scc3-I358ins overexpression.A. Flowchart of the experimental design. B. Strain YIO81 with plasmid pRS406 (pGAL URA3), pIO88 (pGAL-SCC3 URA3) or pIO88R1 (pGAL-scc3-I358ins URA3) 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). C. 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).

Mentions: To assess the molecular defect of RID A insertions, we chose to study the insertion after I358 residue of Scc3 (scc3-I358ins). Given cohesin’s important role in sister chromatid cohesion, we first tested whether scc3-I358ins overexpression induced a cohesion defect. Cultures of haploid strain YIO81 (scc3-6) containing the empty pGAL vector, pGAL-SCC3 or pGAL-scc3-I358ins were arrested in G1 at 23°C using alpha factor. Galactose was added and cells incubated for 1 hour to induce the GAL promoter. Cells were released from G1 at 23°C into YPGAL media containing nocodazole to allow cell-cycle progression under inducing conditions through arrest in G2/M (Fig. 2A). Cohesion was analyzed at the LYS4 locus using the LacI-GFP spot assay (Materials and Methods). In cells carrying either the empty vector or the wild-type SCC3 allele, only about 10% of cells had two GFP spots, indicating robust cohesion at the permissive temperature of 23°C. In contrast, close to 50% of scc3-I358ins expressing cells had 2-GFP signals, indicative of precocious sister chromatid separation (Fig. 2B). Thus, over-expression of scc3-I358ins from G1 to G2/M induced a major cohesion defect.


A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex.

Orgil O, Matityahu A, Eng T, Guacci V, Koshland D, Onn I - PLoS Genet. (2015)

Analysis of sister chromatid cohesion upon scc3-I358ins overexpression.A. Flowchart of the experimental design. B. Strain YIO81 with plasmid pRS406 (pGAL URA3), pIO88 (pGAL-SCC3 URA3) or pIO88R1 (pGAL-scc3-I358ins URA3) 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). C. 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).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352044&req=5

pgen.1005036.g002: Analysis of sister chromatid cohesion upon scc3-I358ins overexpression.A. Flowchart of the experimental design. B. Strain YIO81 with plasmid pRS406 (pGAL URA3), pIO88 (pGAL-SCC3 URA3) or pIO88R1 (pGAL-scc3-I358ins URA3) 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). C. 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).
Mentions: To assess the molecular defect of RID A insertions, we chose to study the insertion after I358 residue of Scc3 (scc3-I358ins). Given cohesin’s important role in sister chromatid cohesion, we first tested whether scc3-I358ins overexpression induced a cohesion defect. Cultures of haploid strain YIO81 (scc3-6) containing the empty pGAL vector, pGAL-SCC3 or pGAL-scc3-I358ins were arrested in G1 at 23°C using alpha factor. Galactose was added and cells incubated for 1 hour to induce the GAL promoter. Cells were released from G1 at 23°C into YPGAL media containing nocodazole to allow cell-cycle progression under inducing conditions through arrest in G2/M (Fig. 2A). Cohesion was analyzed at the LYS4 locus using the LacI-GFP spot assay (Materials and Methods). In cells carrying either the empty vector or the wild-type SCC3 allele, only about 10% of cells had two GFP spots, indicating robust cohesion at the permissive temperature of 23°C. In contrast, close to 50% of scc3-I358ins expressing cells had 2-GFP signals, indicative of precocious sister chromatid separation (Fig. 2B). Thus, over-expression of scc3-I358ins from G1 to G2/M induced a major cohesion defect.

Bottom Line: Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader.These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion.These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medicine in The Galilee, Bar-Ilan University, Safed, Israel.

ABSTRACT
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 SCC3 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.

Show MeSH
Related in: MedlinePlus