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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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hMYH physically interacts with hRad9. (A) HEK293 cells were transfected with plasmids encoding c-myc, c-myc-hMYH-full, or a c-myc-tagged hMYH deletion mutants (−ΔN, −ΔC, or -ΔNC) and FLAG-tagged hRad9, as indicated at the top of the figure. Co-IP was performed with anti-c-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) hRad9 directly interacts with the N-terminus of hMYH. A GST pull-down assay was performed using GST, GST-hMYH-full, or a GST-hMYH deletion mutants (ΔN, ΔC, or ΔNC). Purified His-hRad9 was incubated with GST, GST-hMYH-full, or a GST-hMYH deletion mutants immobilized on glutathione beads. Bound proteins were separated by SDS-PAGE and detected with immunoblotting using anti-GST and anti-His antibodies. Lane 1: purified His-hRad9, lanes 2–6: pull-downs of His-hRad9 with GST (lane 2), GST-hMYH-full (lane 3), GST-hMYH-ΔN (lane 4), GST-hMYH-ΔC (lane 5), and GST-hMYH-ΔNC (lane 6).
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Figure 3: hMYH physically interacts with hRad9. (A) HEK293 cells were transfected with plasmids encoding c-myc, c-myc-hMYH-full, or a c-myc-tagged hMYH deletion mutants (−ΔN, −ΔC, or -ΔNC) and FLAG-tagged hRad9, as indicated at the top of the figure. Co-IP was performed with anti-c-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) hRad9 directly interacts with the N-terminus of hMYH. A GST pull-down assay was performed using GST, GST-hMYH-full, or a GST-hMYH deletion mutants (ΔN, ΔC, or ΔNC). Purified His-hRad9 was incubated with GST, GST-hMYH-full, or a GST-hMYH deletion mutants immobilized on glutathione beads. Bound proteins were separated by SDS-PAGE and detected with immunoblotting using anti-GST and anti-His antibodies. Lane 1: purified His-hRad9, lanes 2–6: pull-downs of His-hRad9 with GST (lane 2), GST-hMYH-full (lane 3), GST-hMYH-ΔN (lane 4), GST-hMYH-ΔC (lane 5), and GST-hMYH-ΔNC (lane 6).

Mentions: Photoswitching activity used to identify Dronpa protein [16]. To evaluate the photoswitching activity of native or complemented Dronpa fragments, HEK293 cells were co-transfected with vectors expressing Dronpa or hMYH-LDN and DCL-hRad9. The cells were incubated for 24 h and analyzed by confocal fluorescence microscopy. After visualization, cells exhibiting Dronpa fluorescence were irradiated at 488 nm for 2 min to induce photobleaching and then photoactivated at 430 nm for 30 s. The similar results were obtained with native and complemented Dronpa fragments (data not shown).To confirm this interaction, we transfected different sets of vectors into HEK293 cells (c-myc, c-myc-hMYH-full, c-myc-hMYH-ΔN, c-myc-hMYH-ΔC, or c-myc-hMYH-ΔNC and FLAG-hRad9). After incubation for 24 h, cell lysates were extracted and immunoprecipitated with anti-c-myc antibody (Figure 3A). Immunoblot analysis was performed with anti-c-myc or anti-FLAG antibody. In agreement with Figure 2C, FLAG-hRad9 precipitated with c-myc-hMYH-full and c-myc-hMYH-ΔC (Figure 3A upper panel). To further demonstrate the direct interaction between hRad9 and hMYH, we performed a GST pull-down assay, as previously described. As same with IP results, Purified His-hRad9 was pulled down by GST-hMYH-full and GST-hMYH-ΔC, but not by GST alone, GST-hMYH-ΔN, and GST-hMYH-ΔNC (Figure 3B). These results indicate that the N-terminal region of hMYH facilitates the interaction between hMYH and hRad9.


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)

hMYH physically interacts with hRad9. (A) HEK293 cells were transfected with plasmids encoding c-myc, c-myc-hMYH-full, or a c-myc-tagged hMYH deletion mutants (−ΔN, −ΔC, or -ΔNC) and FLAG-tagged hRad9, as indicated at the top of the figure. Co-IP was performed with anti-c-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) hRad9 directly interacts with the N-terminus of hMYH. A GST pull-down assay was performed using GST, GST-hMYH-full, or a GST-hMYH deletion mutants (ΔN, ΔC, or ΔNC). Purified His-hRad9 was incubated with GST, GST-hMYH-full, or a GST-hMYH deletion mutants immobilized on glutathione beads. Bound proteins were separated by SDS-PAGE and detected with immunoblotting using anti-GST and anti-His antibodies. Lane 1: purified His-hRad9, lanes 2–6: pull-downs of His-hRad9 with GST (lane 2), GST-hMYH-full (lane 3), GST-hMYH-ΔN (lane 4), GST-hMYH-ΔC (lane 5), and GST-hMYH-ΔNC (lane 6).
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Figure 3: hMYH physically interacts with hRad9. (A) HEK293 cells were transfected with plasmids encoding c-myc, c-myc-hMYH-full, or a c-myc-tagged hMYH deletion mutants (−ΔN, −ΔC, or -ΔNC) and FLAG-tagged hRad9, as indicated at the top of the figure. Co-IP was performed with anti-c-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) hRad9 directly interacts with the N-terminus of hMYH. A GST pull-down assay was performed using GST, GST-hMYH-full, or a GST-hMYH deletion mutants (ΔN, ΔC, or ΔNC). Purified His-hRad9 was incubated with GST, GST-hMYH-full, or a GST-hMYH deletion mutants immobilized on glutathione beads. Bound proteins were separated by SDS-PAGE and detected with immunoblotting using anti-GST and anti-His antibodies. Lane 1: purified His-hRad9, lanes 2–6: pull-downs of His-hRad9 with GST (lane 2), GST-hMYH-full (lane 3), GST-hMYH-ΔN (lane 4), GST-hMYH-ΔC (lane 5), and GST-hMYH-ΔNC (lane 6).
Mentions: Photoswitching activity used to identify Dronpa protein [16]. To evaluate the photoswitching activity of native or complemented Dronpa fragments, HEK293 cells were co-transfected with vectors expressing Dronpa or hMYH-LDN and DCL-hRad9. The cells were incubated for 24 h and analyzed by confocal fluorescence microscopy. After visualization, cells exhibiting Dronpa fluorescence were irradiated at 488 nm for 2 min to induce photobleaching and then photoactivated at 430 nm for 30 s. The similar results were obtained with native and complemented Dronpa fragments (data not shown).To confirm this interaction, we transfected different sets of vectors into HEK293 cells (c-myc, c-myc-hMYH-full, c-myc-hMYH-ΔN, c-myc-hMYH-ΔC, or c-myc-hMYH-ΔNC and FLAG-hRad9). After incubation for 24 h, cell lysates were extracted and immunoprecipitated with anti-c-myc antibody (Figure 3A). Immunoblot analysis was performed with anti-c-myc or anti-FLAG antibody. In agreement with Figure 2C, FLAG-hRad9 precipitated with c-myc-hMYH-full and c-myc-hMYH-ΔC (Figure 3A upper panel). To further demonstrate the direct interaction between hRad9 and hMYH, we performed a GST pull-down assay, as previously described. As same with IP results, Purified His-hRad9 was pulled down by GST-hMYH-full and GST-hMYH-ΔC, but not by GST alone, GST-hMYH-ΔN, and GST-hMYH-ΔNC (Figure 3B). These results indicate that the N-terminal region of hMYH facilitates the interaction between hMYH and hRad9.

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