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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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Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.A. Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (SCC2-3V5), YIO92 (SCC2-3V5 SCC3-6HA) or YIO92R1 (SCC2-3V5 scc3-I358ins-6HA) 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. Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (WPL1-3V5) YOG3007 (WPL1-3V5 SCC3-6HA) or YOG3008 (WPL1-3V5 scc3-I358ins-6HA) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).
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pgen.1005036.g004: Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.A. Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (SCC2-3V5), YIO92 (SCC2-3V5 SCC3-6HA) or YIO92R1 (SCC2-3V5 scc3-I358ins-6HA) 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. Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (WPL1-3V5) YOG3007 (WPL1-3V5 SCC3-6HA) or YOG3008 (WPL1-3V5 scc3-I358ins-6HA) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).

Mentions: The defect in scc3-I358ins allele likely arose from its inability to interact with the cohesin trimer via Mcd1 or one of the cohesin regulators known to bind Scc3, such as Scc2, Pds5 and Wpl1. To test this hypothesis, we examined the ability of Scc3-I358ins-6HA to co-immunoprecipitate with Mcd1, Scc2-3V5, Pds5 and Wpl1-3V5 using appropriate monoclonal or polyclonal antibodies (Fig. 4). We found that scc3-I358ins dramatically reduces the amount of Mcd1 that co-immunoprecipitates as compared to WT Scc3 (Fig. 4A). In contrast, scc3-I358ins and wild-type Scc3 bound Scc2-3V5, Pds5 and Wpl1-3V5 at similar levels (Fig. 4A and B). These analyses suggest that I358, and likely the entire RID A region, is required specifically for Scc3 to bind Mcd1. Furthermore, it corroborates previous in vitro studies that suggested that Scc3 can bind Scc2 and the Wpl1-Pds5 complex independently of its interaction with Mcd1 [24,26]. Finally, it strongly suggests that interaction of Scc3 with Mcd1 is necessary for cohesin loading and the establishment of sister chromatid cohesion.


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)

Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.A. Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (SCC2-3V5), YIO92 (SCC2-3V5 SCC3-6HA) or YIO92R1 (SCC2-3V5 scc3-I358ins-6HA) 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. Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (WPL1-3V5) YOG3007 (WPL1-3V5 SCC3-6HA) or YOG3008 (WPL1-3V5 scc3-I358ins-6HA) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).
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pgen.1005036.g004: Co-immunoprecipitation of Scc3-I358Ins with core and accessory cohesin subunits.A. Analysis of scc3-I358ins-6HA binding to Mcd1, Scc2 and Pds5. Haploids JH5257 (SCC2-3V5), YIO92 (SCC2-3V5 SCC3-6HA) or YIO92R1 (SCC2-3V5 scc3-I358ins-6HA) 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. Analysis of scc3-I358ins-6HA binding to Wpl1. VG3333 (WPL1-3V5) YOG3007 (WPL1-3V5 SCC3-6HA) or YOG3008 (WPL1-3V5 scc3-I358ins-6HA) cells were analyzed as described in A. Precipitated proteins were analyzed by Western blot using antibodies against HA (IP) and V5 (co-IP).
Mentions: The defect in scc3-I358ins allele likely arose from its inability to interact with the cohesin trimer via Mcd1 or one of the cohesin regulators known to bind Scc3, such as Scc2, Pds5 and Wpl1. To test this hypothesis, we examined the ability of Scc3-I358ins-6HA to co-immunoprecipitate with Mcd1, Scc2-3V5, Pds5 and Wpl1-3V5 using appropriate monoclonal or polyclonal antibodies (Fig. 4). We found that scc3-I358ins dramatically reduces the amount of Mcd1 that co-immunoprecipitates as compared to WT Scc3 (Fig. 4A). In contrast, scc3-I358ins and wild-type Scc3 bound Scc2-3V5, Pds5 and Wpl1-3V5 at similar levels (Fig. 4A and B). These analyses suggest that I358, and likely the entire RID A region, is required specifically for Scc3 to bind Mcd1. Furthermore, it corroborates previous in vitro studies that suggested that Scc3 can bind Scc2 and the Wpl1-Pds5 complex independently of its interaction with Mcd1 [24,26]. Finally, it strongly suggests that interaction of Scc3 with Mcd1 is necessary for cohesin loading and the establishment of sister chromatid cohesion.

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