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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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HU induces hMYH and hRad9 expression. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of hMYH and hRad9 mRNA expression. Total RNA was extracted from untreated or treated (HU or H2O2) cells and used for RT-PCR. The levels of hMYH and hRad9 mRNA were determined and normalized to β-actin levels (unpaired t-test; p < 0.05; *significant; **not significant) (B) hRad9 and hMYH expression in HEK293 cells increased after HU treatment, leading to cell cycle arrest at the G1/S phase. Total cell lysates from untreated, HU-treated, or H2O2-treated cells were used for immunoblotting to measure the expression of hMYH, hRad9, ATR, p-Chk1 (S345), Chk1, p-Cdk2 (T14, Y15), and Cdk2 proteins. (C) hRad9 and hMYH knockdown reduced the phosphorylation of Chk1 and Cdk2 in HU-treated cells. HEK293 cells were transfected with siRNA for GFP, hMYH, hRad9, or hMYH and hRad9 as indicated in the figure. Transfected cells were treated with 20 mM HU for 1 h, and then allowed to recover in fresh medium for 2 h. Total cell lysates were used for immunoblotting analysis with anti-hMYH, anti-hRad9, anti-ATR, anti-p-Chk1 (S345), anti-Chk1, anti-p-Cdk2 (T14, Y15), anti-Cdk2, and anti-actin antibodies.
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Figure 4: HU induces hMYH and hRad9 expression. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of hMYH and hRad9 mRNA expression. Total RNA was extracted from untreated or treated (HU or H2O2) cells and used for RT-PCR. The levels of hMYH and hRad9 mRNA were determined and normalized to β-actin levels (unpaired t-test; p < 0.05; *significant; **not significant) (B) hRad9 and hMYH expression in HEK293 cells increased after HU treatment, leading to cell cycle arrest at the G1/S phase. Total cell lysates from untreated, HU-treated, or H2O2-treated cells were used for immunoblotting to measure the expression of hMYH, hRad9, ATR, p-Chk1 (S345), Chk1, p-Cdk2 (T14, Y15), and Cdk2 proteins. (C) hRad9 and hMYH knockdown reduced the phosphorylation of Chk1 and Cdk2 in HU-treated cells. HEK293 cells were transfected with siRNA for GFP, hMYH, hRad9, or hMYH and hRad9 as indicated in the figure. Transfected cells were treated with 20 mM HU for 1 h, and then allowed to recover in fresh medium for 2 h. Total cell lysates were used for immunoblotting analysis with anti-hMYH, anti-hRad9, anti-ATR, anti-p-Chk1 (S345), anti-Chk1, anti-p-Cdk2 (T14, Y15), anti-Cdk2, and anti-actin antibodies.

Mentions: We examined the physiological effects of the hRad9 and hMYH interaction by exposing cells to damaging agents (HU). hMYH and hRad9 mRNA levels were examined by RT-PCR. After normalization to actin, hMYH intensity was higher in HU-treated samples (0.85), compared to the intensity in untreated (0.55) and H2O2-treated (0.60) samples. The same result was observed for hRad9, which increased after HU treatment (0.72 vs. 0.40 in untreated and 0.43 in H2O2-treated). Thus, we noted a marked increase in hRad9 and hMYH mRNA levels after treatment with HU, but not after H2O2 treatment (Figure 4A). Moreover, hMYH and hRad9 protein expression also increased in HU-treated cells, but not in H2O2-treated cells.


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)

HU induces hMYH and hRad9 expression. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of hMYH and hRad9 mRNA expression. Total RNA was extracted from untreated or treated (HU or H2O2) cells and used for RT-PCR. The levels of hMYH and hRad9 mRNA were determined and normalized to β-actin levels (unpaired t-test; p < 0.05; *significant; **not significant) (B) hRad9 and hMYH expression in HEK293 cells increased after HU treatment, leading to cell cycle arrest at the G1/S phase. Total cell lysates from untreated, HU-treated, or H2O2-treated cells were used for immunoblotting to measure the expression of hMYH, hRad9, ATR, p-Chk1 (S345), Chk1, p-Cdk2 (T14, Y15), and Cdk2 proteins. (C) hRad9 and hMYH knockdown reduced the phosphorylation of Chk1 and Cdk2 in HU-treated cells. HEK293 cells were transfected with siRNA for GFP, hMYH, hRad9, or hMYH and hRad9 as indicated in the figure. Transfected cells were treated with 20 mM HU for 1 h, and then allowed to recover in fresh medium for 2 h. Total cell lysates were used for immunoblotting analysis with anti-hMYH, anti-hRad9, anti-ATR, anti-p-Chk1 (S345), anti-Chk1, anti-p-Cdk2 (T14, Y15), anti-Cdk2, and anti-actin antibodies.
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Figure 4: HU induces hMYH and hRad9 expression. (A) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of hMYH and hRad9 mRNA expression. Total RNA was extracted from untreated or treated (HU or H2O2) cells and used for RT-PCR. The levels of hMYH and hRad9 mRNA were determined and normalized to β-actin levels (unpaired t-test; p < 0.05; *significant; **not significant) (B) hRad9 and hMYH expression in HEK293 cells increased after HU treatment, leading to cell cycle arrest at the G1/S phase. Total cell lysates from untreated, HU-treated, or H2O2-treated cells were used for immunoblotting to measure the expression of hMYH, hRad9, ATR, p-Chk1 (S345), Chk1, p-Cdk2 (T14, Y15), and Cdk2 proteins. (C) hRad9 and hMYH knockdown reduced the phosphorylation of Chk1 and Cdk2 in HU-treated cells. HEK293 cells were transfected with siRNA for GFP, hMYH, hRad9, or hMYH and hRad9 as indicated in the figure. Transfected cells were treated with 20 mM HU for 1 h, and then allowed to recover in fresh medium for 2 h. Total cell lysates were used for immunoblotting analysis with anti-hMYH, anti-hRad9, anti-ATR, anti-p-Chk1 (S345), anti-Chk1, anti-p-Cdk2 (T14, Y15), anti-Cdk2, and anti-actin antibodies.
Mentions: We examined the physiological effects of the hRad9 and hMYH interaction by exposing cells to damaging agents (HU). hMYH and hRad9 mRNA levels were examined by RT-PCR. After normalization to actin, hMYH intensity was higher in HU-treated samples (0.85), compared to the intensity in untreated (0.55) and H2O2-treated (0.60) samples. The same result was observed for hRad9, which increased after HU treatment (0.72 vs. 0.40 in untreated and 0.43 in H2O2-treated). Thus, we noted a marked increase in hRad9 and hMYH mRNA levels after treatment with HU, but not after H2O2 treatment (Figure 4A). Moreover, hMYH and hRad9 protein expression also increased in HU-treated cells, but not in H2O2-treated cells.

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