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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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Rad21–Rad21 interaction determined by yeast two-hybrid assay. (A) Expression of Rad21 wild type (1–631 aa), CT-L (254–631 aa), and CT (451–631 aa) in yeast was analyzed with WBs using Rad21 CT–specific pAb. (B) A LacZ reporter assay was used to probe the Rad21–Rad21 interaction. Yeast cells cotransfected with pC97 and pC86 empty vector is shown as the negative control. (C) pC97 GAL4BD and pC86 GAL4AD were used as the positive control. Positive protein–protein interaction is shown in blue in th LacZ reporter assay.
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fig5: Rad21–Rad21 interaction determined by yeast two-hybrid assay. (A) Expression of Rad21 wild type (1–631 aa), CT-L (254–631 aa), and CT (451–631 aa) in yeast was analyzed with WBs using Rad21 CT–specific pAb. (B) A LacZ reporter assay was used to probe the Rad21–Rad21 interaction. Yeast cells cotransfected with pC97 and pC86 empty vector is shown as the negative control. (C) pC97 GAL4BD and pC86 GAL4AD were used as the positive control. Positive protein–protein interaction is shown in blue in th LacZ reporter assay.

Mentions: In our previous yeast two-hybrid assay using Rad21 NT long (NT-L; 1–283 aa) and CT long (CT-L; 254–631 aa) as bait, we isolated full-length Rad21 as an interactor (unpublished data). To further verify the interaction of Rad21–Rad21 shown in the aforementioned co-IP and PCA studies, we extended our yeast two-hybrid assay as additional evidence. The cDNAs of full-length Rad21, Rad21 NT-L, Rad21 CT-L, and Rad21 CT (450–631 aa) were cloned into GAL4 DNA–binding (pC97) and GAL4 DNA activation (pC86) domain plasmids. WB results show that the constructs are well expressed in the two-hybrid yeast stain MV103 (Fig. 5 A). A yeast two-hybrid assay demonstrates that full-length Rad21, Rad21 NT-L, and Rad21 CT-L interact with themselves and with each other (Fig. 5 B). However, NT-truncated Rad21 (Rad21 CT) failed to interact with itself or with other Rad21 constructs (Fig. 5 B), which is consistent with the IP results (not depicted). Based on the findings in PCA and yeast two-hybrid assay, we conclude that Rad21 interacts with Rad21 and aligns in an antiparallel manner in the cohesin complex.


A handcuff model for the cohesin complex.

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

Rad21–Rad21 interaction determined by yeast two-hybrid assay. (A) Expression of Rad21 wild type (1–631 aa), CT-L (254–631 aa), and CT (451–631 aa) in yeast was analyzed with WBs using Rad21 CT–specific pAb. (B) A LacZ reporter assay was used to probe the Rad21–Rad21 interaction. Yeast cells cotransfected with pC97 and pC86 empty vector is shown as the negative control. (C) pC97 GAL4BD and pC86 GAL4AD were used as the positive control. Positive protein–protein interaction is shown in blue in th LacZ reporter assay.
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600748&req=5

fig5: Rad21–Rad21 interaction determined by yeast two-hybrid assay. (A) Expression of Rad21 wild type (1–631 aa), CT-L (254–631 aa), and CT (451–631 aa) in yeast was analyzed with WBs using Rad21 CT–specific pAb. (B) A LacZ reporter assay was used to probe the Rad21–Rad21 interaction. Yeast cells cotransfected with pC97 and pC86 empty vector is shown as the negative control. (C) pC97 GAL4BD and pC86 GAL4AD were used as the positive control. Positive protein–protein interaction is shown in blue in th LacZ reporter assay.
Mentions: In our previous yeast two-hybrid assay using Rad21 NT long (NT-L; 1–283 aa) and CT long (CT-L; 254–631 aa) as bait, we isolated full-length Rad21 as an interactor (unpublished data). To further verify the interaction of Rad21–Rad21 shown in the aforementioned co-IP and PCA studies, we extended our yeast two-hybrid assay as additional evidence. The cDNAs of full-length Rad21, Rad21 NT-L, Rad21 CT-L, and Rad21 CT (450–631 aa) were cloned into GAL4 DNA–binding (pC97) and GAL4 DNA activation (pC86) domain plasmids. WB results show that the constructs are well expressed in the two-hybrid yeast stain MV103 (Fig. 5 A). A yeast two-hybrid assay demonstrates that full-length Rad21, Rad21 NT-L, and Rad21 CT-L interact with themselves and with each other (Fig. 5 B). However, NT-truncated Rad21 (Rad21 CT) failed to interact with itself or with other Rad21 constructs (Fig. 5 B), which is consistent with the IP results (not depicted). Based on the findings in PCA and yeast two-hybrid assay, we conclude that Rad21 interacts with Rad21 and aligns in an antiparallel manner in the cohesin complex.

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