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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 Y371 binding to the rDNA is reduced.Strains YOG3021 (SCC3-6HA scc3-6) and YOG3024 (scc3-Y371A-6HA scc3-6) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: A. chromosome III CARC1; B. chromosome IV centromere; C. chromosoXII rDNA. A representative PCR is shown (n = 3).
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pgen.1005036.g009: scc3 Y371 binding to the rDNA is reduced.Strains YOG3021 (SCC3-6HA scc3-6) and YOG3024 (scc3-Y371A-6HA scc3-6) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: A. chromosome III CARC1; B. chromosome IV centromere; C. chromosoXII rDNA. A representative PCR is shown (n = 3).

Mentions: We next addressed what was the molecular defect in scc3-Y371A that allowed condensation but not cohesion? Given that this allele lay within a region that we showed was necessary for cohesin loading on chromosomes (between I358 and D373), it seemed likely that this allele also affected cohesin binding to chromosomes. Since scc3-Y371A was cohesion proficient, at least some cohesin must be loaded onto chromosomes. Therefore we anticipated that scc3-Y371A would reduce rather than abrogate cohesin binding to chromosomes. To test this possibility, we performed a ChIP assays by immunoprecipating the Scc3 Y371A-6HA protein and assaying its localization at CARs, centromeric DNA and the rDNA (Fig. 9). Cohesin binding on CARC1 on chromosome III was comparable to the wild type cells. Strikingly, about 2–3 fold reduction in cohesin binding to centromere of chromosome IV was found in scc3-Y371A cells as compared to wild type cells. Similar reduction was observed in the rDNA locus. Thus, the region encompassing RID A and SCD not only contains residues that controls global binding of cohesin to chromosomes, but also contains at least one residue (Y371) that controls binding to specific regions. The reduced loading of cohesin specifically at the centromere and rDNA in scc3-Y371A is consistent with a condensation defect as these regions appear to have unique condensation requirements as reflected by their robust recruitment of condensin [41,42].


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 Y371 binding to the rDNA is reduced.Strains YOG3021 (SCC3-6HA scc3-6) and YOG3024 (scc3-Y371A-6HA scc3-6) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: A. chromosome III CARC1; B. chromosome IV centromere; C. chromosoXII rDNA. A representative PCR is shown (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005036.g009: scc3 Y371 binding to the rDNA is reduced.Strains YOG3021 (SCC3-6HA scc3-6) and YOG3024 (scc3-Y371A-6HA scc3-6) were processed for chromatin immunoprecipitation analysis. Scc3 was immunoprecipitated with anti-HA antibodies and the precipitated DNA was analyzed by quantitative PCR for: A. chromosome III CARC1; B. chromosome IV centromere; C. chromosoXII rDNA. A representative PCR is shown (n = 3).
Mentions: We next addressed what was the molecular defect in scc3-Y371A that allowed condensation but not cohesion? Given that this allele lay within a region that we showed was necessary for cohesin loading on chromosomes (between I358 and D373), it seemed likely that this allele also affected cohesin binding to chromosomes. Since scc3-Y371A was cohesion proficient, at least some cohesin must be loaded onto chromosomes. Therefore we anticipated that scc3-Y371A would reduce rather than abrogate cohesin binding to chromosomes. To test this possibility, we performed a ChIP assays by immunoprecipating the Scc3 Y371A-6HA protein and assaying its localization at CARs, centromeric DNA and the rDNA (Fig. 9). Cohesin binding on CARC1 on chromosome III was comparable to the wild type cells. Strikingly, about 2–3 fold reduction in cohesin binding to centromere of chromosome IV was found in scc3-Y371A cells as compared to wild type cells. Similar reduction was observed in the rDNA locus. Thus, the region encompassing RID A and SCD not only contains residues that controls global binding of cohesin to chromosomes, but also contains at least one residue (Y371) that controls binding to specific regions. The reduced loading of cohesin specifically at the centromere and rDNA in scc3-Y371A is consistent with a condensation defect as these regions appear to have unique condensation requirements as reflected by their robust recruitment of condensin [41,42].

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