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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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Co-IP and WB analysis of cohesin Rad21–Rad21 interaction. Logarithmically growing 293T or HeLa cells were transfected with appropriate Rad21 plasmids or empty vector (EV). Input (10% of IP) and IP samples were resolved by 7% SDS-PAGE and blotted with the indicated anti-bodies. (A) Flag-Rad21 and Myc-Rad21 coimmunoprecipitated. (B) Myc-Rad21 coimmunoprecipitated endogenous Rad21. Bar graphs show the relative level of Myc-Rad21 and endogenous Rad21 in input (left) and co-IP samples (right). Error bars indicate SEM from three observations. (C) Radom primer PCR amplification of DNA from 293T cell lysates and the elutes of immunoprecipitated samples. The template DNA used for PCR was purified from cell lysates after nuclease treatment as shown in lanes 1–4. The cell lysates for IP with Flag mAb (lanes 5 and 6) or Myc pAb (lanes 7 and 8) agarose-conjugated beads were digested with DNase I and RNase A in lanes 5 and 7 but were not digested in lanes 6 and 8. The amounts of DNA template used for PCR were from 125 μg of protein of cell lysates (lanes 1–4) or 1 mg of protein of cell lysates in IP samples (lanes 5–8). There is no DNA in the negative control (lane 9) and 0.25 ng DNA in the positive control (lane 10). (D) Cell lysates were treated with or without DNase I and RNase A before co-IP. (E) Co-IP of Flag-Rad21 and Myc-Rad21 from cells in which Flag-Rad21 and Myc-Rad21 were expressed separately, and lysates were mixed together before IP was performed (**, lanes 6 and 13) or from cells cotransfected with Flag-Rad21 and Myc-Rad21 (lanes 7 and 14). Black lines indicate that intervening lanes have been spliced out.
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fig1: Co-IP and WB analysis of cohesin Rad21–Rad21 interaction. Logarithmically growing 293T or HeLa cells were transfected with appropriate Rad21 plasmids or empty vector (EV). Input (10% of IP) and IP samples were resolved by 7% SDS-PAGE and blotted with the indicated anti-bodies. (A) Flag-Rad21 and Myc-Rad21 coimmunoprecipitated. (B) Myc-Rad21 coimmunoprecipitated endogenous Rad21. Bar graphs show the relative level of Myc-Rad21 and endogenous Rad21 in input (left) and co-IP samples (right). Error bars indicate SEM from three observations. (C) Radom primer PCR amplification of DNA from 293T cell lysates and the elutes of immunoprecipitated samples. The template DNA used for PCR was purified from cell lysates after nuclease treatment as shown in lanes 1–4. The cell lysates for IP with Flag mAb (lanes 5 and 6) or Myc pAb (lanes 7 and 8) agarose-conjugated beads were digested with DNase I and RNase A in lanes 5 and 7 but were not digested in lanes 6 and 8. The amounts of DNA template used for PCR were from 125 μg of protein of cell lysates (lanes 1–4) or 1 mg of protein of cell lysates in IP samples (lanes 5–8). There is no DNA in the negative control (lane 9) and 0.25 ng DNA in the positive control (lane 10). (D) Cell lysates were treated with or without DNase I and RNase A before co-IP. (E) Co-IP of Flag-Rad21 and Myc-Rad21 from cells in which Flag-Rad21 and Myc-Rad21 were expressed separately, and lysates were mixed together before IP was performed (**, lanes 6 and 13) or from cells cotransfected with Flag-Rad21 and Myc-Rad21 (lanes 7 and 14). Black lines indicate that intervening lanes have been spliced out.

Mentions: To investigate whether more than one Rad21 protein is in the cohesin complex, we coexpressed two Rad21 constructs with different epitopes in 293T cells, coimmunoprecipitated the protein, and analyzed the results using WB. The results indicate that Flag-Rad21 coimmunoprecipitates Myc-Rad21 and vice versa (Fig. 1 A). Moreover, WB with Rad21 pAb indicates that equal amounts of Myc-Rad21 and Flag-Rad21/endogenous Rad21 is coimmunoprecipitated (Fig. 1 A, right). It is possible that Myc-Rad21 forms a dimer with Flag-Rad21/endogenous Rad21 when Myc-Rad21 is underexpressed compared with Flag-Rad21/endogenous Rad21 (see next paragraph).


A handcuff model for the cohesin complex.

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

Co-IP and WB analysis of cohesin Rad21–Rad21 interaction. Logarithmically growing 293T or HeLa cells were transfected with appropriate Rad21 plasmids or empty vector (EV). Input (10% of IP) and IP samples were resolved by 7% SDS-PAGE and blotted with the indicated anti-bodies. (A) Flag-Rad21 and Myc-Rad21 coimmunoprecipitated. (B) Myc-Rad21 coimmunoprecipitated endogenous Rad21. Bar graphs show the relative level of Myc-Rad21 and endogenous Rad21 in input (left) and co-IP samples (right). Error bars indicate SEM from three observations. (C) Radom primer PCR amplification of DNA from 293T cell lysates and the elutes of immunoprecipitated samples. The template DNA used for PCR was purified from cell lysates after nuclease treatment as shown in lanes 1–4. The cell lysates for IP with Flag mAb (lanes 5 and 6) or Myc pAb (lanes 7 and 8) agarose-conjugated beads were digested with DNase I and RNase A in lanes 5 and 7 but were not digested in lanes 6 and 8. The amounts of DNA template used for PCR were from 125 μg of protein of cell lysates (lanes 1–4) or 1 mg of protein of cell lysates in IP samples (lanes 5–8). There is no DNA in the negative control (lane 9) and 0.25 ng DNA in the positive control (lane 10). (D) Cell lysates were treated with or without DNase I and RNase A before co-IP. (E) Co-IP of Flag-Rad21 and Myc-Rad21 from cells in which Flag-Rad21 and Myc-Rad21 were expressed separately, and lysates were mixed together before IP was performed (**, lanes 6 and 13) or from cells cotransfected with Flag-Rad21 and Myc-Rad21 (lanes 7 and 14). Black lines indicate that intervening lanes have been spliced out.
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fig1: Co-IP and WB analysis of cohesin Rad21–Rad21 interaction. Logarithmically growing 293T or HeLa cells were transfected with appropriate Rad21 plasmids or empty vector (EV). Input (10% of IP) and IP samples were resolved by 7% SDS-PAGE and blotted with the indicated anti-bodies. (A) Flag-Rad21 and Myc-Rad21 coimmunoprecipitated. (B) Myc-Rad21 coimmunoprecipitated endogenous Rad21. Bar graphs show the relative level of Myc-Rad21 and endogenous Rad21 in input (left) and co-IP samples (right). Error bars indicate SEM from three observations. (C) Radom primer PCR amplification of DNA from 293T cell lysates and the elutes of immunoprecipitated samples. The template DNA used for PCR was purified from cell lysates after nuclease treatment as shown in lanes 1–4. The cell lysates for IP with Flag mAb (lanes 5 and 6) or Myc pAb (lanes 7 and 8) agarose-conjugated beads were digested with DNase I and RNase A in lanes 5 and 7 but were not digested in lanes 6 and 8. The amounts of DNA template used for PCR were from 125 μg of protein of cell lysates (lanes 1–4) or 1 mg of protein of cell lysates in IP samples (lanes 5–8). There is no DNA in the negative control (lane 9) and 0.25 ng DNA in the positive control (lane 10). (D) Cell lysates were treated with or without DNase I and RNase A before co-IP. (E) Co-IP of Flag-Rad21 and Myc-Rad21 from cells in which Flag-Rad21 and Myc-Rad21 were expressed separately, and lysates were mixed together before IP was performed (**, lanes 6 and 13) or from cells cotransfected with Flag-Rad21 and Myc-Rad21 (lanes 7 and 14). Black lines indicate that intervening lanes have been spliced out.
Mentions: To investigate whether more than one Rad21 protein is in the cohesin complex, we coexpressed two Rad21 constructs with different epitopes in 293T cells, coimmunoprecipitated the protein, and analyzed the results using WB. The results indicate that Flag-Rad21 coimmunoprecipitates Myc-Rad21 and vice versa (Fig. 1 A). Moreover, WB with Rad21 pAb indicates that equal amounts of Myc-Rad21 and Flag-Rad21/endogenous Rad21 is coimmunoprecipitated (Fig. 1 A, right). It is possible that Myc-Rad21 forms a dimer with Flag-Rad21/endogenous Rad21 when Myc-Rad21 is underexpressed compared with Flag-Rad21/endogenous Rad21 (see next paragraph).

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