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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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hRad9 interacts with the N-terminal region of hMYH. (A) Vector for the Dronpa-BiFC system used to identify the interacting region of hMYH. Expression vectors were created as previously described; however, the N-terminus of Dronpa (DN, 1–164) was C-terminally fused to either full-length, wild-type hMYH or mutant hMYH [ΔN, ΔC, ΔNC]. The C-terminal region of Dronpa (DC, 165–224) was N-terminally fused to hRad9. Dronpa and DCL-hRad9 were tagged with FLAG; hMYH-full-LDN and the deletion mutants were tagged with c-myc. (B) The expression of each protein in transfected HEK293 cells was detected by immunoblotting with anti-FLAG and anti-c-myc antibodies. (C) The Dronpa-BiFC system demonstrated that the N-terminal region of hMYH is important for the interaction between hMYH and hRad9. HEK293 cells were seeded on a cover glass-bottom dish at a density of 1 × 105 cells per well. Cells were transfected with plasmids encoding full-length hMYH-LDN or a deletion mutant (hMYH-ΔN-LDN, hMYH-ΔC-LDN, or hMYH-ΔNC-LDN) and DCL-Rad9, as indicated to the left of the figure and incubated for 24 h. Fluorescence was assessed using a confocal fluorescence microscope with 488-nm excitation and 530-nm emission filters. Scale bar, 50 μm.
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Figure 2: hRad9 interacts with the N-terminal region of hMYH. (A) Vector for the Dronpa-BiFC system used to identify the interacting region of hMYH. Expression vectors were created as previously described; however, the N-terminus of Dronpa (DN, 1–164) was C-terminally fused to either full-length, wild-type hMYH or mutant hMYH [ΔN, ΔC, ΔNC]. The C-terminal region of Dronpa (DC, 165–224) was N-terminally fused to hRad9. Dronpa and DCL-hRad9 were tagged with FLAG; hMYH-full-LDN and the deletion mutants were tagged with c-myc. (B) The expression of each protein in transfected HEK293 cells was detected by immunoblotting with anti-FLAG and anti-c-myc antibodies. (C) The Dronpa-BiFC system demonstrated that the N-terminal region of hMYH is important for the interaction between hMYH and hRad9. HEK293 cells were seeded on a cover glass-bottom dish at a density of 1 × 105 cells per well. Cells were transfected with plasmids encoding full-length hMYH-LDN or a deletion mutant (hMYH-ΔN-LDN, hMYH-ΔC-LDN, or hMYH-ΔNC-LDN) and DCL-Rad9, as indicated to the left of the figure and incubated for 24 h. Fluorescence was assessed using a confocal fluorescence microscope with 488-nm excitation and 530-nm emission filters. Scale bar, 50 μm.

Mentions: The N-terminal region of hMYH contains sites for RPA binding (amino acids 1–32) and function in substrate specificity, and the C-terminal region is important for glycosylase activity [19]. We created hMYH mutants with N-, C-, or N- and C-terminal deletions [ΔN (amino acids 75–547), ΔC (amino acids 1–487), and ΔNC (amino acids 75–487), respectively] to determine the functional impact of the hMYH and hRad9 interaction on substrate specificity and glycosylase activity. We successfully used the Dronpa-BiFC system developed in our lab to identify the region of hMYH that interacts with hRad9 and to visualize the interaction between hMYH-hHus1. The optimum fragments for Dronpa cleavage were selected according to a structural analysis of GFP and mRFP1. BiFC vectors for visualization of the interaction between hRad9 and hMYH were constructed as described in the Methods section [16]. The Dronpa C-terminus was fused to hRad9, and the Dronpa N-terminus was fused to either full-length hMYH (pcDNA3-c-myc/hMYH-LDN) or hMYH deletion mutants (pcDNA3-c-myc/hMYH∆N-LDN, pcDNA3-c-myc/hMYH-∆C-LDN, pcDNA3-c-myc/hMYH-∆NC-LDN). The vector used for the Dronpa-BiFC system is shown in Figure 2A. The transient expression of transfected proteins in cells was confirmed by immunoblotting (Figure 2B).HEK293 cells were transfected with different vector sets of plasmids (Dronpa-full, hMYH-LDN/DCL-hRad9, hMYH-∆N-LDN/DCL-hRad9, hMYH-∆C-LDN/DCL-hRad9, and hMYH-∆NC-LDN/DCL-hRad9). Cells were incubated for 24 h, and Dronpa fluorescence was visualized by fluorescence microscopy. Dronpa fluorescence was observed in cells transfected with native Dronpa and in cells co-transfected with hMYH-LDN/DCL-hRad9 and hMYH-∆C-LDN/DCL-hRad9, but no fluorescence was observed in cells transfected with hMYH-∆N-LDN/DCL-hRad9 and hMYH-∆NC-LDN/DCL-hRad9 (Figure 2C). These results showed that hRad9 interacts with the N-terminal region of hMYH. The Dronpa fluorescence observed in cells transfected with DCL-hRad9 and hMYH-LDN or hMYH-∆C-LDN was due to reconstitution of functional Dronpa facilitated by the interaction between hRad9 and hMYH.


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)

hRad9 interacts with the N-terminal region of hMYH. (A) Vector for the Dronpa-BiFC system used to identify the interacting region of hMYH. Expression vectors were created as previously described; however, the N-terminus of Dronpa (DN, 1–164) was C-terminally fused to either full-length, wild-type hMYH or mutant hMYH [ΔN, ΔC, ΔNC]. The C-terminal region of Dronpa (DC, 165–224) was N-terminally fused to hRad9. Dronpa and DCL-hRad9 were tagged with FLAG; hMYH-full-LDN and the deletion mutants were tagged with c-myc. (B) The expression of each protein in transfected HEK293 cells was detected by immunoblotting with anti-FLAG and anti-c-myc antibodies. (C) The Dronpa-BiFC system demonstrated that the N-terminal region of hMYH is important for the interaction between hMYH and hRad9. HEK293 cells were seeded on a cover glass-bottom dish at a density of 1 × 105 cells per well. Cells were transfected with plasmids encoding full-length hMYH-LDN or a deletion mutant (hMYH-ΔN-LDN, hMYH-ΔC-LDN, or hMYH-ΔNC-LDN) and DCL-Rad9, as indicated to the left of the figure and incubated for 24 h. Fluorescence was assessed using a confocal fluorescence microscope with 488-nm excitation and 530-nm emission filters. Scale bar, 50 μm.
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Related In: Results  -  Collection

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Figure 2: hRad9 interacts with the N-terminal region of hMYH. (A) Vector for the Dronpa-BiFC system used to identify the interacting region of hMYH. Expression vectors were created as previously described; however, the N-terminus of Dronpa (DN, 1–164) was C-terminally fused to either full-length, wild-type hMYH or mutant hMYH [ΔN, ΔC, ΔNC]. The C-terminal region of Dronpa (DC, 165–224) was N-terminally fused to hRad9. Dronpa and DCL-hRad9 were tagged with FLAG; hMYH-full-LDN and the deletion mutants were tagged with c-myc. (B) The expression of each protein in transfected HEK293 cells was detected by immunoblotting with anti-FLAG and anti-c-myc antibodies. (C) The Dronpa-BiFC system demonstrated that the N-terminal region of hMYH is important for the interaction between hMYH and hRad9. HEK293 cells were seeded on a cover glass-bottom dish at a density of 1 × 105 cells per well. Cells were transfected with plasmids encoding full-length hMYH-LDN or a deletion mutant (hMYH-ΔN-LDN, hMYH-ΔC-LDN, or hMYH-ΔNC-LDN) and DCL-Rad9, as indicated to the left of the figure and incubated for 24 h. Fluorescence was assessed using a confocal fluorescence microscope with 488-nm excitation and 530-nm emission filters. Scale bar, 50 μm.
Mentions: The N-terminal region of hMYH contains sites for RPA binding (amino acids 1–32) and function in substrate specificity, and the C-terminal region is important for glycosylase activity [19]. We created hMYH mutants with N-, C-, or N- and C-terminal deletions [ΔN (amino acids 75–547), ΔC (amino acids 1–487), and ΔNC (amino acids 75–487), respectively] to determine the functional impact of the hMYH and hRad9 interaction on substrate specificity and glycosylase activity. We successfully used the Dronpa-BiFC system developed in our lab to identify the region of hMYH that interacts with hRad9 and to visualize the interaction between hMYH-hHus1. The optimum fragments for Dronpa cleavage were selected according to a structural analysis of GFP and mRFP1. BiFC vectors for visualization of the interaction between hRad9 and hMYH were constructed as described in the Methods section [16]. The Dronpa C-terminus was fused to hRad9, and the Dronpa N-terminus was fused to either full-length hMYH (pcDNA3-c-myc/hMYH-LDN) or hMYH deletion mutants (pcDNA3-c-myc/hMYH∆N-LDN, pcDNA3-c-myc/hMYH-∆C-LDN, pcDNA3-c-myc/hMYH-∆NC-LDN). The vector used for the Dronpa-BiFC system is shown in Figure 2A. The transient expression of transfected proteins in cells was confirmed by immunoblotting (Figure 2B).HEK293 cells were transfected with different vector sets of plasmids (Dronpa-full, hMYH-LDN/DCL-hRad9, hMYH-∆N-LDN/DCL-hRad9, hMYH-∆C-LDN/DCL-hRad9, and hMYH-∆NC-LDN/DCL-hRad9). Cells were incubated for 24 h, and Dronpa fluorescence was visualized by fluorescence microscopy. Dronpa fluorescence was observed in cells transfected with native Dronpa and in cells co-transfected with hMYH-LDN/DCL-hRad9 and hMYH-∆C-LDN/DCL-hRad9, but no fluorescence was observed in cells transfected with hMYH-∆N-LDN/DCL-hRad9 and hMYH-∆NC-LDN/DCL-hRad9 (Figure 2C). These results showed that hRad9 interacts with the N-terminal region of hMYH. The Dronpa fluorescence observed in cells transfected with DCL-hRad9 and hMYH-LDN or hMYH-∆C-LDN was due to reconstitution of functional Dronpa facilitated by the interaction between hRad9 and hMYH.

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