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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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Each cohesin complex contains only one copy of SA1 or SA2. 293T cells were cotransfected with the respective constructs. 48 h after transfection, IP was performed. SA1 and SA2 were detected using the corresponding antibodies shown on the blots. (A) HA and Flag epitopes were tagged to the NT of SA1 and SA2. (B and C) HA and Myc epitopes were tagged to the CT of SA1 and SA2. EV, empty vector; *, splicing SA1-Myc or SA2-Myc; **, nonspecific band. Black lines indicate that intervening lanes have been spliced out.
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fig3: Each cohesin complex contains only one copy of SA1 or SA2. 293T cells were cotransfected with the respective constructs. 48 h after transfection, IP was performed. SA1 and SA2 were detected using the corresponding antibodies shown on the blots. (A) HA and Flag epitopes were tagged to the NT of SA1 and SA2. (B and C) HA and Myc epitopes were tagged to the CT of SA1 and SA2. EV, empty vector; *, splicing SA1-Myc or SA2-Myc; **, nonspecific band. Black lines indicate that intervening lanes have been spliced out.

Mentions: In budding yeast, Scc3 is a core subunit of the cohesin complex. In humans, there are two orthologues of Scc3, SA1 and SA2, and SA2 is more abundant than SA1 (Losada et al., 2000). Because three of the four cohesin core subunits, Rad21, Smc1, and Smc3, can immunoprecipitate themselves, the cohesin complex might contain more than one copy of the fourth core subunit, SA1/SA2. To investigate whether SA1 can immunoprecipitate itself, we cotransfected 293T cells with Flag-SA1 and HA-SA1 constructs. An IP experiment using asynchronous 293T cells demonstrates that Flag-SA1 and HA-SA1 cannot coimmunoprecipitate (Fig. 3 A, lanes 5 and 11). The inability to detect an interaction between Flag-SA1 and HA-SA1 was not caused by failure of the IP, as Flag and HA antisera efficiently detected the bands of Flag-SA1 and HA-SA1, respectively (Fig. 3 A). We also obtained similar results when Flag-SA1 and Myc-SA1 were used in co-IP experiments (unpublished data). Similar to SA1, neither Flag-SA2 or HA-SA2 (Fig. 3 A, lanes 6 and 12) nor Myc-SA2 or Flag-SA2 (not depicted) can immunoprecipitate each other.


A handcuff model for the cohesin complex.

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

Each cohesin complex contains only one copy of SA1 or SA2. 293T cells were cotransfected with the respective constructs. 48 h after transfection, IP was performed. SA1 and SA2 were detected using the corresponding antibodies shown on the blots. (A) HA and Flag epitopes were tagged to the NT of SA1 and SA2. (B and C) HA and Myc epitopes were tagged to the CT of SA1 and SA2. EV, empty vector; *, splicing SA1-Myc or SA2-Myc; **, nonspecific band. Black lines indicate that intervening lanes have been spliced out.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Each cohesin complex contains only one copy of SA1 or SA2. 293T cells were cotransfected with the respective constructs. 48 h after transfection, IP was performed. SA1 and SA2 were detected using the corresponding antibodies shown on the blots. (A) HA and Flag epitopes were tagged to the NT of SA1 and SA2. (B and C) HA and Myc epitopes were tagged to the CT of SA1 and SA2. EV, empty vector; *, splicing SA1-Myc or SA2-Myc; **, nonspecific band. Black lines indicate that intervening lanes have been spliced out.
Mentions: In budding yeast, Scc3 is a core subunit of the cohesin complex. In humans, there are two orthologues of Scc3, SA1 and SA2, and SA2 is more abundant than SA1 (Losada et al., 2000). Because three of the four cohesin core subunits, Rad21, Smc1, and Smc3, can immunoprecipitate themselves, the cohesin complex might contain more than one copy of the fourth core subunit, SA1/SA2. To investigate whether SA1 can immunoprecipitate itself, we cotransfected 293T cells with Flag-SA1 and HA-SA1 constructs. An IP experiment using asynchronous 293T cells demonstrates that Flag-SA1 and HA-SA1 cannot coimmunoprecipitate (Fig. 3 A, lanes 5 and 11). The inability to detect an interaction between Flag-SA1 and HA-SA1 was not caused by failure of the IP, as Flag and HA antisera efficiently detected the bands of Flag-SA1 and HA-SA1, respectively (Fig. 3 A). We also obtained similar results when Flag-SA1 and Myc-SA1 were used in co-IP experiments (unpublished data). Similar to SA1, neither Flag-SA2 or HA-SA2 (Fig. 3 A, lanes 6 and 12) nor Myc-SA2 or Flag-SA2 (not depicted) can immunoprecipitate each other.

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