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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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Cohesion and chromosome binding of scc3-I358Ins under native expression levels.A. Strains YIO81 (scc3-6), YIO91 (SCC3-6HA scc3-6) andYIO91R1 (scc3-I358ins-6HA scc3-6) 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 scc3-6. B. Flowchart of the experimental design to score sister chromatid cohesion. C. 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 (S1 Fig). D. 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). E. 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).
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pgen.1005036.g003: Cohesion and chromosome binding of scc3-I358Ins under native expression levels.A. Strains YIO81 (scc3-6), YIO91 (SCC3-6HA scc3-6) andYIO91R1 (scc3-I358ins-6HA scc3-6) 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 scc3-6. B. Flowchart of the experimental design to score sister chromatid cohesion. C. 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 (S1 Fig). D. 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). E. 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).

Mentions: To further dissect the molecular defect of scc3-I358ins, we generated SCC3-6HA and scc3-I358ins-6HA alleles, which contain six hemagglutinin epitopes (6HA) at the C-terminus, and are under control of the endogenous SCC3 promoter. These alleles were integrated into haploid strain YIO81 (scc3-6) at the URA3 locus. The YIO81 parent alone or bearing either SCC3-6HA or scc3-I358ins-6HA were grown to saturation, serially diluted on YPD plates and incubated at 23°C or 37°C, the permissive or restrictive temperature of the scc3-6 allele, respectively (Fig. 3A). At 23°C, the scc3-6 strain alone and the scc3-I358ins-6HA scc3-6 strain grew equally well, but slightly slower than SCC3-6HA scc3-6 strain. Thus the scc3-6 allele is slightly compromised for Scc3 function at the permissive temperature, but even so, endogenous expression levels of scc3-I358ins-6HA is recessive in contrast to its dominant-negative phenotype when overexpressed (Figs. 1C and 3A). At 37°C, the scc3-6 strain alone and the scc3-I358ins-6HA scc3-6 strain fail to grow whereas the SCC3-6HA scc3-6 strain is viable.


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)

Cohesion and chromosome binding of scc3-I358Ins under native expression levels.A. Strains YIO81 (scc3-6), YIO91 (SCC3-6HA scc3-6) andYIO91R1 (scc3-I358ins-6HA scc3-6) 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 scc3-6. B. Flowchart of the experimental design to score sister chromatid cohesion. C. 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 (S1 Fig). D. 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). E. 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).
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pgen.1005036.g003: Cohesion and chromosome binding of scc3-I358Ins under native expression levels.A. Strains YIO81 (scc3-6), YIO91 (SCC3-6HA scc3-6) andYIO91R1 (scc3-I358ins-6HA scc3-6) 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 scc3-6. B. Flowchart of the experimental design to score sister chromatid cohesion. C. 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 (S1 Fig). D. 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). E. 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).
Mentions: To further dissect the molecular defect of scc3-I358ins, we generated SCC3-6HA and scc3-I358ins-6HA alleles, which contain six hemagglutinin epitopes (6HA) at the C-terminus, and are under control of the endogenous SCC3 promoter. These alleles were integrated into haploid strain YIO81 (scc3-6) at the URA3 locus. The YIO81 parent alone or bearing either SCC3-6HA or scc3-I358ins-6HA were grown to saturation, serially diluted on YPD plates and incubated at 23°C or 37°C, the permissive or restrictive temperature of the scc3-6 allele, respectively (Fig. 3A). At 23°C, the scc3-6 strain alone and the scc3-I358ins-6HA scc3-6 strain grew equally well, but slightly slower than SCC3-6HA scc3-6 strain. Thus the scc3-6 allele is slightly compromised for Scc3 function at the permissive temperature, but even so, endogenous expression levels of scc3-I358ins-6HA is recessive in contrast to its dominant-negative phenotype when overexpressed (Figs. 1C and 3A). At 37°C, the scc3-6 strain alone and the scc3-I358ins-6HA scc3-6 strain fail to grow whereas the SCC3-6HA scc3-6 strain is viable.

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