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Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation.

Agustina L, Hahm SH, Han SH, Tran AH, Chung JH, Park JH, Park JW, Han YS - BMC Mol. Biol. (2014)

Bottom Line: But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells.This interaction is enhanced by HU treatment.Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Advanced Technology Fusion, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea. yshan@konkuk.ac.kr.

ABSTRACT

Background: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction.

Results: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells.

Conclusions: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.

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Interaction between hMYH and hRad9. (A) HEK293 cells were transfected with c-myc and FLAG-tagged hRad9, or c-myc-tagged hMYH and FLAG-tagged-hRad9 as indicated. Co-IP was performed with anti-c-myc antibody, and the immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) Lysates from untransfected HEK293 cells were used for the immunoprecipitation of hMYH with an anti-hMYH antibody. Immunoblotting was performed using anti-hMYH and anti-hRad9 antibodies. Lane 1: input sample, lanes 2 and 3: proteins immunoprecipitated with mouse IgG or anti-hMYH antibody. (C) HEK293 cells were transfected with siHus1 or siGFP. Total cell lysates were used for co-IP with hRad9 antibody. (D) Pull-down of purified His-hRad9 with GST-hMYH bound to glutathione-Sepharose beads. Lane 1: purified His-hRad9, lanes 2 and 3: pull-downs with GST and GST-hMYH, respectively.
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Figure 1: Interaction between hMYH and hRad9. (A) HEK293 cells were transfected with c-myc and FLAG-tagged hRad9, or c-myc-tagged hMYH and FLAG-tagged-hRad9 as indicated. Co-IP was performed with anti-c-myc antibody, and the immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) Lysates from untransfected HEK293 cells were used for the immunoprecipitation of hMYH with an anti-hMYH antibody. Immunoblotting was performed using anti-hMYH and anti-hRad9 antibodies. Lane 1: input sample, lanes 2 and 3: proteins immunoprecipitated with mouse IgG or anti-hMYH antibody. (C) HEK293 cells were transfected with siHus1 or siGFP. Total cell lysates were used for co-IP with hRad9 antibody. (D) Pull-down of purified His-hRad9 with GST-hMYH bound to glutathione-Sepharose beads. Lane 1: purified His-hRad9, lanes 2 and 3: pull-downs with GST and GST-hMYH, respectively.

Mentions: To determine whether hRad9 interacts with hMYH, co-immunoprecipitation (co-IP) was performed using HEK293 cells transfected with c-myc and FLAG-tagged hRad9 or c-myc-tagged hMYH and FLAG-tagged hRad9. After incubation for 24 h, cell lysates were extracted and immunoprecipitated with an anti-c-myc antibody. Precipitated samples were analyzed by immunoblotting with anti-FLAG and anti-c-myc antibodies. FLAG-hRad9 was precipitated through its interaction with c-myc-hMYH (Figure 1A). In western blot analysis, hRad9 was visualized as several bands due to differences in the phosphorylation states. These results may indicate a physical interaction between hMYH and hRad9. The endogenous interaction between hRad9 and hMYH was confirmed by co-IP with anti-hMYH antibody. hRad9 immunoprecipitated with hMYH (Figure 1B), indicating that endogenous hMYH interacts with hRad9.To determine the involvement of the 9-1-1 complex in the interaction between hMYH and hRad9, Hus1 expression was knocked down using specific siRNA. In Hus1-depleted cells, the interaction between hMYH and hRad9 was decreased (Figure 1C). This result indicates that hRad9 interacts with hMYH as part of the 9-1-1 complex.


Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation.

Agustina L, Hahm SH, Han SH, Tran AH, Chung JH, Park JH, Park JW, Han YS - BMC Mol. Biol. (2014)

Interaction between hMYH and hRad9. (A) HEK293 cells were transfected with c-myc and FLAG-tagged hRad9, or c-myc-tagged hMYH and FLAG-tagged-hRad9 as indicated. Co-IP was performed with anti-c-myc antibody, and the immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) Lysates from untransfected HEK293 cells were used for the immunoprecipitation of hMYH with an anti-hMYH antibody. Immunoblotting was performed using anti-hMYH and anti-hRad9 antibodies. Lane 1: input sample, lanes 2 and 3: proteins immunoprecipitated with mouse IgG or anti-hMYH antibody. (C) HEK293 cells were transfected with siHus1 or siGFP. Total cell lysates were used for co-IP with hRad9 antibody. (D) Pull-down of purified His-hRad9 with GST-hMYH bound to glutathione-Sepharose beads. Lane 1: purified His-hRad9, lanes 2 and 3: pull-downs with GST and GST-hMYH, respectively.
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Related In: Results  -  Collection

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Figure 1: Interaction between hMYH and hRad9. (A) HEK293 cells were transfected with c-myc and FLAG-tagged hRad9, or c-myc-tagged hMYH and FLAG-tagged-hRad9 as indicated. Co-IP was performed with anti-c-myc antibody, and the immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) Lysates from untransfected HEK293 cells were used for the immunoprecipitation of hMYH with an anti-hMYH antibody. Immunoblotting was performed using anti-hMYH and anti-hRad9 antibodies. Lane 1: input sample, lanes 2 and 3: proteins immunoprecipitated with mouse IgG or anti-hMYH antibody. (C) HEK293 cells were transfected with siHus1 or siGFP. Total cell lysates were used for co-IP with hRad9 antibody. (D) Pull-down of purified His-hRad9 with GST-hMYH bound to glutathione-Sepharose beads. Lane 1: purified His-hRad9, lanes 2 and 3: pull-downs with GST and GST-hMYH, respectively.
Mentions: To determine whether hRad9 interacts with hMYH, co-immunoprecipitation (co-IP) was performed using HEK293 cells transfected with c-myc and FLAG-tagged hRad9 or c-myc-tagged hMYH and FLAG-tagged hRad9. After incubation for 24 h, cell lysates were extracted and immunoprecipitated with an anti-c-myc antibody. Precipitated samples were analyzed by immunoblotting with anti-FLAG and anti-c-myc antibodies. FLAG-hRad9 was precipitated through its interaction with c-myc-hMYH (Figure 1A). In western blot analysis, hRad9 was visualized as several bands due to differences in the phosphorylation states. These results may indicate a physical interaction between hMYH and hRad9. The endogenous interaction between hRad9 and hMYH was confirmed by co-IP with anti-hMYH antibody. hRad9 immunoprecipitated with hMYH (Figure 1B), indicating that endogenous hMYH interacts with hRad9.To determine the involvement of the 9-1-1 complex in the interaction between hMYH and hRad9, Hus1 expression was knocked down using specific siRNA. In Hus1-depleted cells, the interaction between hMYH and hRad9 was decreased (Figure 1C). This result indicates that hRad9 interacts with hMYH as part of the 9-1-1 complex.

Bottom Line: But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells.This interaction is enhanced by HU treatment.Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Advanced Technology Fusion, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea. yshan@konkuk.ac.kr.

ABSTRACT

Background: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction.

Results: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells.

Conclusions: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.

Show MeSH
Related in: MedlinePlus