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A handcuff model for the cohesin complex.

Zhang N, Kuznetsov SG, Sharan SK, Li K, Rao PH, Pati D - J. Cell Biol. (2008)

Bottom Line: Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field.The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner.These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The cohesin complex is responsible for the accurate separation of sister chromatids into two daughter cells. Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field. However, the static configuration of the embrace model is not flexible enough for cohesins to perform their functions during DNA replication, transcription, and DNA repair. We used coimmunoprecipitation, a protein fragment complement assay, and a yeast two-hybrid assay to analyze the protein-protein interactions among cohesin subunits. The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner. These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

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Fluorescent protein fragment complementation assay showing the Rad21–Rad21 interaction and the antiparallel orientation. (A) YFP(NT) or YFP(CT) were fused to either the NT or CT end of Rad21. YFP-fused Rad21 constructs were expressed in 293T cells (lanes 3–6), and their interaction with the cohesin complex was examined by IP of the endogenous Smc3 using rabbit anti-Smc3 antisera (lanes 9–12). *, nonspecific band. (B) 293T cells were cotransfected with YFP(NT)- and YFP(CT)-fused Rad21 plasmids (a total of four combinations). YFP fluorescence was examined under a fluorescent microscope 40 h after transfection. (C) YFP fluorescence–positive 293T and HeLa cells transfected with the combination of YFP(NT)-Rad21 and Rad21-YFP(CT) at 400× magnification. (D) Possible antiparallel orientation of Rad21–Rad21 interactions. Only the combination of plasmids in the top panel results in the fluorescence. Bars, 25 μm.
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fig4: Fluorescent protein fragment complementation assay showing the Rad21–Rad21 interaction and the antiparallel orientation. (A) YFP(NT) or YFP(CT) were fused to either the NT or CT end of Rad21. YFP-fused Rad21 constructs were expressed in 293T cells (lanes 3–6), and their interaction with the cohesin complex was examined by IP of the endogenous Smc3 using rabbit anti-Smc3 antisera (lanes 9–12). *, nonspecific band. (B) 293T cells were cotransfected with YFP(NT)- and YFP(CT)-fused Rad21 plasmids (a total of four combinations). YFP fluorescence was examined under a fluorescent microscope 40 h after transfection. (C) YFP fluorescence–positive 293T and HeLa cells transfected with the combination of YFP(NT)-Rad21 and Rad21-YFP(CT) at 400× magnification. (D) Possible antiparallel orientation of Rad21–Rad21 interactions. Only the combination of plasmids in the top panel results in the fluorescence. Bars, 25 μm.

Mentions: Using Smc3 pAb, we tested the expression of the YFP fragment–fused Rad21 molecules in 293T cells and their incorporation into the cohesin complex by immunoprecipitating endogenous Smc3 (Fig. 4 A). All YFP-fused Rad21 are expressed (Fig. 4 A). YFP(CT)-Rad21 could not be detected as efficiently by the GFP pAb as the other three YFP-fused Rad21. However, the level of YFP(CT)-Rad21 is found to be equivalent to endogenous Rad21 when the blot was probed with Rad21 mAb (Fig. 4 A, lane 4). IP of endogenous Smc3 using Smc3 pAb not only efficiently coimmunoprecipitated all four YFP-fused Rad21 proteins but also coimmunoprecipitated endogenous Rad21 (Fig. 4 A, lanes 9–12) along with the other cohesin subunits Smc1, SA1, and SA2 (Fig. 4 A). The four YFP-tagged Rad21 and the other three cohesin core subunits, Smc1, Smc3, and SA1/2, can also be immunoprecipitated by GFP pAb (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200801157/DC1). These data suggest that exogenously YFP-fused Rad21 can be incorporated into the cohesin complex as endogenous Rad21.


A handcuff model for the cohesin complex.

Zhang N, Kuznetsov SG, Sharan SK, Li K, Rao PH, Pati D - J. Cell Biol. (2008)

Fluorescent protein fragment complementation assay showing the Rad21–Rad21 interaction and the antiparallel orientation. (A) YFP(NT) or YFP(CT) were fused to either the NT or CT end of Rad21. YFP-fused Rad21 constructs were expressed in 293T cells (lanes 3–6), and their interaction with the cohesin complex was examined by IP of the endogenous Smc3 using rabbit anti-Smc3 antisera (lanes 9–12). *, nonspecific band. (B) 293T cells were cotransfected with YFP(NT)- and YFP(CT)-fused Rad21 plasmids (a total of four combinations). YFP fluorescence was examined under a fluorescent microscope 40 h after transfection. (C) YFP fluorescence–positive 293T and HeLa cells transfected with the combination of YFP(NT)-Rad21 and Rad21-YFP(CT) at 400× magnification. (D) Possible antiparallel orientation of Rad21–Rad21 interactions. Only the combination of plasmids in the top panel results in the fluorescence. Bars, 25 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2600748&req=5

fig4: Fluorescent protein fragment complementation assay showing the Rad21–Rad21 interaction and the antiparallel orientation. (A) YFP(NT) or YFP(CT) were fused to either the NT or CT end of Rad21. YFP-fused Rad21 constructs were expressed in 293T cells (lanes 3–6), and their interaction with the cohesin complex was examined by IP of the endogenous Smc3 using rabbit anti-Smc3 antisera (lanes 9–12). *, nonspecific band. (B) 293T cells were cotransfected with YFP(NT)- and YFP(CT)-fused Rad21 plasmids (a total of four combinations). YFP fluorescence was examined under a fluorescent microscope 40 h after transfection. (C) YFP fluorescence–positive 293T and HeLa cells transfected with the combination of YFP(NT)-Rad21 and Rad21-YFP(CT) at 400× magnification. (D) Possible antiparallel orientation of Rad21–Rad21 interactions. Only the combination of plasmids in the top panel results in the fluorescence. Bars, 25 μm.
Mentions: Using Smc3 pAb, we tested the expression of the YFP fragment–fused Rad21 molecules in 293T cells and their incorporation into the cohesin complex by immunoprecipitating endogenous Smc3 (Fig. 4 A). All YFP-fused Rad21 are expressed (Fig. 4 A). YFP(CT)-Rad21 could not be detected as efficiently by the GFP pAb as the other three YFP-fused Rad21. However, the level of YFP(CT)-Rad21 is found to be equivalent to endogenous Rad21 when the blot was probed with Rad21 mAb (Fig. 4 A, lane 4). IP of endogenous Smc3 using Smc3 pAb not only efficiently coimmunoprecipitated all four YFP-fused Rad21 proteins but also coimmunoprecipitated endogenous Rad21 (Fig. 4 A, lanes 9–12) along with the other cohesin subunits Smc1, SA1, and SA2 (Fig. 4 A). The four YFP-tagged Rad21 and the other three cohesin core subunits, Smc1, Smc3, and SA1/2, can also be immunoprecipitated by GFP pAb (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200801157/DC1). These data suggest that exogenously YFP-fused Rad21 can be incorporated into the cohesin complex as endogenous Rad21.

Bottom Line: Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field.The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner.These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The cohesin complex is responsible for the accurate separation of sister chromatids into two daughter cells. Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field. However, the static configuration of the embrace model is not flexible enough for cohesins to perform their functions during DNA replication, transcription, and DNA repair. We used coimmunoprecipitation, a protein fragment complement assay, and a yeast two-hybrid assay to analyze the protein-protein interactions among cohesin subunits. The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner. These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

Show MeSH
Related in: MedlinePlus