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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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DNA damage (HU) promotes the interaction between hRad9 and hMYH. (A) Fluorescence resonance energy transfer (FRET) was used to analyze the interaction between over-expressed hRad9 and hMYH after DNA damage. Cells transfected with ECFP and EYFP or ECFP/hMYH and EYFP/hRad9 were treated with or without HU. ECFP, EYFP, and FRET fluorescence was observed with a fluorescence microscope at 440/480 nm (ECFP), 480/535 nm (EYFP), and 440/535 nm (FRET). Scale bar, 50 μm. (B) FRET efficiency was used to quantify the interaction between hMYH and hRad9 after DNA damage. HEK293 cells were transfected with ECFP and EYFP or ECFP/hRad9 and EYFP/hMYH as indicated to the left of the figure. Cells were treated with or without HU. ECFP, EYFP, and FRET fluorescence were measured at the indicated wavelengths. FRET efficiency was quantified in five control cells and five HU-treated cells. Standard error is shown. Scale bar, 10 μm. (C) Immunofluorescence of endogenous hMYH and hRad9. Cells were treated with or without 20 mM HU for 1 h and allowed to recover for 2 h. Cells were stained with antibodies against hMYH (FPG 456, green), hRad9 (FPR 552, yellow), and To-pro®-3 (nuclei, red). Scale bar, 10 μm.
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Figure 5: DNA damage (HU) promotes the interaction between hRad9 and hMYH. (A) Fluorescence resonance energy transfer (FRET) was used to analyze the interaction between over-expressed hRad9 and hMYH after DNA damage. Cells transfected with ECFP and EYFP or ECFP/hMYH and EYFP/hRad9 were treated with or without HU. ECFP, EYFP, and FRET fluorescence was observed with a fluorescence microscope at 440/480 nm (ECFP), 480/535 nm (EYFP), and 440/535 nm (FRET). Scale bar, 50 μm. (B) FRET efficiency was used to quantify the interaction between hMYH and hRad9 after DNA damage. HEK293 cells were transfected with ECFP and EYFP or ECFP/hRad9 and EYFP/hMYH as indicated to the left of the figure. Cells were treated with or without HU. ECFP, EYFP, and FRET fluorescence were measured at the indicated wavelengths. FRET efficiency was quantified in five control cells and five HU-treated cells. Standard error is shown. Scale bar, 10 μm. (C) Immunofluorescence of endogenous hMYH and hRad9. Cells were treated with or without 20 mM HU for 1 h and allowed to recover for 2 h. Cells were stained with antibodies against hMYH (FPG 456, green), hRad9 (FPR 552, yellow), and To-pro®-3 (nuclei, red). Scale bar, 10 μm.

Mentions: The 9-1-1 complex and hMYH participate in DNA damage repair [2,4]. The interaction between hRad9 and hMYH after HU treatment was examined by FRET analysis. HEK293 cells were transfected with ECFP/hMYH and EYFP/hRad9, individually or together. Using confocal fluorescence microscopy, we detected ECFP and EYFP fluorescence in all transfected cells. FRET fluorescence was only observed in cells co-transfected with ECFP/hMYH and EYFP/hRad9. Moreover, higher intensity FRET fluorescence was seen in cells treated with HU, suggesting that HU treatment enhances the interaction between hRad9 and hMYH (Figure 5A).The interaction between hRad9 and hMYH exhibited a relatively high FRET intensity (Figure 5B). Given this result, we determined whether endogenous hMYH and hRad9 localized to the same nuclear foci after HU treatment. In immunofluorescence analysis, hMYH and hRad9 staining in the nucleus of untreated cells was faint (Figure 5C). However, in HU-treated cells, hMYH and hRad9 formed discrete nuclear foci, and a significant fraction of the hMYH nuclear foci co-localized with hRad9, indicating that hMYH and the 9-1-1 complex translocated to the same lesions after DNA damage.


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)

DNA damage (HU) promotes the interaction between hRad9 and hMYH. (A) Fluorescence resonance energy transfer (FRET) was used to analyze the interaction between over-expressed hRad9 and hMYH after DNA damage. Cells transfected with ECFP and EYFP or ECFP/hMYH and EYFP/hRad9 were treated with or without HU. ECFP, EYFP, and FRET fluorescence was observed with a fluorescence microscope at 440/480 nm (ECFP), 480/535 nm (EYFP), and 440/535 nm (FRET). Scale bar, 50 μm. (B) FRET efficiency was used to quantify the interaction between hMYH and hRad9 after DNA damage. HEK293 cells were transfected with ECFP and EYFP or ECFP/hRad9 and EYFP/hMYH as indicated to the left of the figure. Cells were treated with or without HU. ECFP, EYFP, and FRET fluorescence were measured at the indicated wavelengths. FRET efficiency was quantified in five control cells and five HU-treated cells. Standard error is shown. Scale bar, 10 μm. (C) Immunofluorescence of endogenous hMYH and hRad9. Cells were treated with or without 20 mM HU for 1 h and allowed to recover for 2 h. Cells were stained with antibodies against hMYH (FPG 456, green), hRad9 (FPR 552, yellow), and To-pro®-3 (nuclei, red). Scale bar, 10 μm.
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Figure 5: DNA damage (HU) promotes the interaction between hRad9 and hMYH. (A) Fluorescence resonance energy transfer (FRET) was used to analyze the interaction between over-expressed hRad9 and hMYH after DNA damage. Cells transfected with ECFP and EYFP or ECFP/hMYH and EYFP/hRad9 were treated with or without HU. ECFP, EYFP, and FRET fluorescence was observed with a fluorescence microscope at 440/480 nm (ECFP), 480/535 nm (EYFP), and 440/535 nm (FRET). Scale bar, 50 μm. (B) FRET efficiency was used to quantify the interaction between hMYH and hRad9 after DNA damage. HEK293 cells were transfected with ECFP and EYFP or ECFP/hRad9 and EYFP/hMYH as indicated to the left of the figure. Cells were treated with or without HU. ECFP, EYFP, and FRET fluorescence were measured at the indicated wavelengths. FRET efficiency was quantified in five control cells and five HU-treated cells. Standard error is shown. Scale bar, 10 μm. (C) Immunofluorescence of endogenous hMYH and hRad9. Cells were treated with or without 20 mM HU for 1 h and allowed to recover for 2 h. Cells were stained with antibodies against hMYH (FPG 456, green), hRad9 (FPR 552, yellow), and To-pro®-3 (nuclei, red). Scale bar, 10 μm.
Mentions: The 9-1-1 complex and hMYH participate in DNA damage repair [2,4]. The interaction between hRad9 and hMYH after HU treatment was examined by FRET analysis. HEK293 cells were transfected with ECFP/hMYH and EYFP/hRad9, individually or together. Using confocal fluorescence microscopy, we detected ECFP and EYFP fluorescence in all transfected cells. FRET fluorescence was only observed in cells co-transfected with ECFP/hMYH and EYFP/hRad9. Moreover, higher intensity FRET fluorescence was seen in cells treated with HU, suggesting that HU treatment enhances the interaction between hRad9 and hMYH (Figure 5A).The interaction between hRad9 and hMYH exhibited a relatively high FRET intensity (Figure 5B). Given this result, we determined whether endogenous hMYH and hRad9 localized to the same nuclear foci after HU treatment. In immunofluorescence analysis, hMYH and hRad9 staining in the nucleus of untreated cells was faint (Figure 5C). However, in HU-treated cells, hMYH and hRad9 formed discrete nuclear foci, and a significant fraction of the hMYH nuclear foci co-localized with hRad9, indicating that hMYH and the 9-1-1 complex translocated to the same lesions after DNA damage.

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