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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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Scc3 does not affect the Smc3-Mcd1 interaction.A. Strain YOG3049 (SCC3-AID-V5 SMC3-6HA) 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. Strain YOG3027 (SCC3-AID-V5 SCC2-12MYC) 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.
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pgen.1005036.g006: Scc3 does not affect the Smc3-Mcd1 interaction.A. Strain YOG3049 (SCC3-AID-V5 SMC3-6HA) 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. Strain YOG3027 (SCC3-AID-V5 SCC2-12MYC) 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.

Mentions: These results raise the possibility that Scc3 binds to Scc2-Scc4, and then Scc3 interaction with Mcd1 recruits the loader to the cohesin trimer for interactions with other cohesin subunit. If so, scc3-I358ins, which is unable to bind Mcd1, could sequester Scc2-Scc4 away from the trimer and thereby prevent cohesin from binding chromosomes. To test this model we first asked whether Mcd1 can still interact with the Smc subunits in the absence of Scc3. Many experiments have shown that the trimer can form independently of Scc3 but only in vitro. To remove Scc3 in vivo, we generated an auxin-induced degron of Scc3, SCC3-3V5-AID [38,39]. SCC3-3V5-AID strains also contained an Smc3 allele that was tagged internally with a 6HA epitope. SCC3-3V5-AID SMC3-6HA cells were grown to mid-log phase then Scc3-3V5-AID was depleted by addition of 1 mM IAA and incubation for 1 hour. Western blot analysis showed that Scc3-3V5-AID was depleted to undetectable levels (Fig. 6A, left side). We immunoprecipitated Smc3-6HA and assayed its interaction with Mcd1. Smc3 co-immunoprecipitation with Mcd1 was not affected by depletion of Scc3 (Fig. 6A). We infer from this result that Scc3 is not essential for the formation of the Smc1-Smc3-Mcd1 trimer, corroborating the previous in vitro studies.


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)

Scc3 does not affect the Smc3-Mcd1 interaction.A. Strain YOG3049 (SCC3-AID-V5 SMC3-6HA) 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. Strain YOG3027 (SCC3-AID-V5 SCC2-12MYC) 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352044&req=5

pgen.1005036.g006: Scc3 does not affect the Smc3-Mcd1 interaction.A. Strain YOG3049 (SCC3-AID-V5 SMC3-6HA) 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. Strain YOG3027 (SCC3-AID-V5 SCC2-12MYC) 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.
Mentions: These results raise the possibility that Scc3 binds to Scc2-Scc4, and then Scc3 interaction with Mcd1 recruits the loader to the cohesin trimer for interactions with other cohesin subunit. If so, scc3-I358ins, which is unable to bind Mcd1, could sequester Scc2-Scc4 away from the trimer and thereby prevent cohesin from binding chromosomes. To test this model we first asked whether Mcd1 can still interact with the Smc subunits in the absence of Scc3. Many experiments have shown that the trimer can form independently of Scc3 but only in vitro. To remove Scc3 in vivo, we generated an auxin-induced degron of Scc3, SCC3-3V5-AID [38,39]. SCC3-3V5-AID strains also contained an Smc3 allele that was tagged internally with a 6HA epitope. SCC3-3V5-AID SMC3-6HA cells were grown to mid-log phase then Scc3-3V5-AID was depleted by addition of 1 mM IAA and incubation for 1 hour. Western blot analysis showed that Scc3-3V5-AID was depleted to undetectable levels (Fig. 6A, left side). We immunoprecipitated Smc3-6HA and assayed its interaction with Mcd1. Smc3 co-immunoprecipitation with Mcd1 was not affected by depletion of Scc3 (Fig. 6A). We infer from this result that Scc3 is not essential for the formation of the Smc1-Smc3-Mcd1 trimer, corroborating the previous in vitro studies.

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