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JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks.

Wang S, Gong Z, Chen R, Liu Y, Li A, Li G, Zhou J - Nucleic Acids Res. (2009)

Bottom Line: Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide.On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1.In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Toxicology, Cancer Centre, School of Public Health, Nanjing Medical University, Nanjing 210029, People's Republic of China.

ABSTRACT
JWA was recently demonstrated to be involved in cellular responses to environmental stress including oxidative stress. Although it was found that JWA protected cells from reactive oxygen species-induced DNA damage, upregulated base excision repair (BER) protein XRCC1 and downregulated PARP-1, the molecular mechanism of JWA in regulating the repair of DNA single-strand breaks (SSBs) is still unclear. Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide. JWA functioned as a repair protein by multi-interaction with XRCC1. On the one hand, JWA was translocated into the nucleus by the carrier protein XRCC1 and co-localized with XRCC1 foci after oxidative DNA damage. On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1. In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome. These findings indicate that JWA may serve as a novel regulator of XRCC1 in the BER protein complex to facilitate the repair of DNA SSBs.

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JWA is required for repairing H2O2-damaged DNA. (A) The repair efficiency of NIH-3T3 cells on damaged plasmid DNA was detected by HCR assay. NIH-3T3 cells were transfected with control LUC plasmid DNA (pGL3-control) or the same plasmid damaged by hydrogen peroxide at concentrations of 1, 3, 10 or 20% (v/v). DNA repair rates were measured as the ratio of LUC activity in extracts from cells transfected with a damaged plasmid to the LUC activity in extracts from cells transfected with an undamaged plasmid. The experiment was done in triplicate. (B) NIH-3T3 cells were transiently transfected with plasmids to knockdown JWA (JWA shRNA), or with the corresponding control vector (Ctrl shRNA), or were left untreated. After 48 h, whole-cell lysates were collected for detection of target proteins by immunoblotting. (C) NIH-3T3 cells were transiently transfected with a plasmid expressing human JWA protein (EGFP-JWA) or a control vector (EGFP-C1). Expression of target proteins 48 h after transfection was examined in whole-cell lysates by immunoblotting. (D) Knockdown of JWA inhibits DRC of damaged plasmids, while overexpression of JWA enhances DRC in NIH-3T3 cells. NIH-3T3 cells were transfected with either the EGFP-C1 control or EGFP-JWA plasmid, control shRNA or JWA shRNA together with undamaged or 10% (v/v) H2O2-damaged LUC plasmids. The pGL3 (undamaged or H2O2-damaged) plamids were transfected as a control for co-transfection efficiency. Twenty-four hours after transfection, the DRC of the damaged LUC reporter was assayed as indicated in (A). The renilla luciferase reporter (internal control, Promega) was used to normalize the activity of the LUC reporter. *P < 0.05.
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Figure 1: JWA is required for repairing H2O2-damaged DNA. (A) The repair efficiency of NIH-3T3 cells on damaged plasmid DNA was detected by HCR assay. NIH-3T3 cells were transfected with control LUC plasmid DNA (pGL3-control) or the same plasmid damaged by hydrogen peroxide at concentrations of 1, 3, 10 or 20% (v/v). DNA repair rates were measured as the ratio of LUC activity in extracts from cells transfected with a damaged plasmid to the LUC activity in extracts from cells transfected with an undamaged plasmid. The experiment was done in triplicate. (B) NIH-3T3 cells were transiently transfected with plasmids to knockdown JWA (JWA shRNA), or with the corresponding control vector (Ctrl shRNA), or were left untreated. After 48 h, whole-cell lysates were collected for detection of target proteins by immunoblotting. (C) NIH-3T3 cells were transiently transfected with a plasmid expressing human JWA protein (EGFP-JWA) or a control vector (EGFP-C1). Expression of target proteins 48 h after transfection was examined in whole-cell lysates by immunoblotting. (D) Knockdown of JWA inhibits DRC of damaged plasmids, while overexpression of JWA enhances DRC in NIH-3T3 cells. NIH-3T3 cells were transfected with either the EGFP-C1 control or EGFP-JWA plasmid, control shRNA or JWA shRNA together with undamaged or 10% (v/v) H2O2-damaged LUC plasmids. The pGL3 (undamaged or H2O2-damaged) plamids were transfected as a control for co-transfection efficiency. Twenty-four hours after transfection, the DRC of the damaged LUC reporter was assayed as indicated in (A). The renilla luciferase reporter (internal control, Promega) was used to normalize the activity of the LUC reporter. *P < 0.05.

Mentions: Studies were undertaken to determine if endogenous JWA plays a physiological role in the repair of H2O2-induced DNA lesions. The HCR assay, which was previously validated in a study of BER proteins and H2O2-induced DNA lesions (44), was conducted in this study. First, a dose-repair model for the LUCcon plasmid was performed, and then the DRC was determined for the plasmids damaged by H2O2. The DRC was reduced to ∼55% for 10% (v/v) H2O2-treated LUCcon plasmids compared to undamaged control plasmids (Figure 1A). In NIH-3T3 cells, the JWA protein level was significantly reduced by ∼97% after transient transfection with JWA shRNA (Figure 1B). Subsequently, the effect of JWA in NIH-3T3 cells was examined by HCR assay, and it was found that the DRC was reduced by more than 80% in JWA-knockdown cells compared to cells transfected with the control vector (Figure 1D). In contrast, overexpression of JWA in the NIH-3T3 cells markedly increased the DRC by up to 2-fold (Figure 1C and D).Figure 1.


JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA single-strand breaks.

Wang S, Gong Z, Chen R, Liu Y, Li A, Li G, Zhou J - Nucleic Acids Res. (2009)

JWA is required for repairing H2O2-damaged DNA. (A) The repair efficiency of NIH-3T3 cells on damaged plasmid DNA was detected by HCR assay. NIH-3T3 cells were transfected with control LUC plasmid DNA (pGL3-control) or the same plasmid damaged by hydrogen peroxide at concentrations of 1, 3, 10 or 20% (v/v). DNA repair rates were measured as the ratio of LUC activity in extracts from cells transfected with a damaged plasmid to the LUC activity in extracts from cells transfected with an undamaged plasmid. The experiment was done in triplicate. (B) NIH-3T3 cells were transiently transfected with plasmids to knockdown JWA (JWA shRNA), or with the corresponding control vector (Ctrl shRNA), or were left untreated. After 48 h, whole-cell lysates were collected for detection of target proteins by immunoblotting. (C) NIH-3T3 cells were transiently transfected with a plasmid expressing human JWA protein (EGFP-JWA) or a control vector (EGFP-C1). Expression of target proteins 48 h after transfection was examined in whole-cell lysates by immunoblotting. (D) Knockdown of JWA inhibits DRC of damaged plasmids, while overexpression of JWA enhances DRC in NIH-3T3 cells. NIH-3T3 cells were transfected with either the EGFP-C1 control or EGFP-JWA plasmid, control shRNA or JWA shRNA together with undamaged or 10% (v/v) H2O2-damaged LUC plasmids. The pGL3 (undamaged or H2O2-damaged) plamids were transfected as a control for co-transfection efficiency. Twenty-four hours after transfection, the DRC of the damaged LUC reporter was assayed as indicated in (A). The renilla luciferase reporter (internal control, Promega) was used to normalize the activity of the LUC reporter. *P < 0.05.
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Figure 1: JWA is required for repairing H2O2-damaged DNA. (A) The repair efficiency of NIH-3T3 cells on damaged plasmid DNA was detected by HCR assay. NIH-3T3 cells were transfected with control LUC plasmid DNA (pGL3-control) or the same plasmid damaged by hydrogen peroxide at concentrations of 1, 3, 10 or 20% (v/v). DNA repair rates were measured as the ratio of LUC activity in extracts from cells transfected with a damaged plasmid to the LUC activity in extracts from cells transfected with an undamaged plasmid. The experiment was done in triplicate. (B) NIH-3T3 cells were transiently transfected with plasmids to knockdown JWA (JWA shRNA), or with the corresponding control vector (Ctrl shRNA), or were left untreated. After 48 h, whole-cell lysates were collected for detection of target proteins by immunoblotting. (C) NIH-3T3 cells were transiently transfected with a plasmid expressing human JWA protein (EGFP-JWA) or a control vector (EGFP-C1). Expression of target proteins 48 h after transfection was examined in whole-cell lysates by immunoblotting. (D) Knockdown of JWA inhibits DRC of damaged plasmids, while overexpression of JWA enhances DRC in NIH-3T3 cells. NIH-3T3 cells were transfected with either the EGFP-C1 control or EGFP-JWA plasmid, control shRNA or JWA shRNA together with undamaged or 10% (v/v) H2O2-damaged LUC plasmids. The pGL3 (undamaged or H2O2-damaged) plamids were transfected as a control for co-transfection efficiency. Twenty-four hours after transfection, the DRC of the damaged LUC reporter was assayed as indicated in (A). The renilla luciferase reporter (internal control, Promega) was used to normalize the activity of the LUC reporter. *P < 0.05.
Mentions: Studies were undertaken to determine if endogenous JWA plays a physiological role in the repair of H2O2-induced DNA lesions. The HCR assay, which was previously validated in a study of BER proteins and H2O2-induced DNA lesions (44), was conducted in this study. First, a dose-repair model for the LUCcon plasmid was performed, and then the DRC was determined for the plasmids damaged by H2O2. The DRC was reduced to ∼55% for 10% (v/v) H2O2-treated LUCcon plasmids compared to undamaged control plasmids (Figure 1A). In NIH-3T3 cells, the JWA protein level was significantly reduced by ∼97% after transient transfection with JWA shRNA (Figure 1B). Subsequently, the effect of JWA in NIH-3T3 cells was examined by HCR assay, and it was found that the DRC was reduced by more than 80% in JWA-knockdown cells compared to cells transfected with the control vector (Figure 1D). In contrast, overexpression of JWA in the NIH-3T3 cells markedly increased the DRC by up to 2-fold (Figure 1C and D).Figure 1.

Bottom Line: Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide.On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1.In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology and Toxicology, Cancer Centre, School of Public Health, Nanjing Medical University, Nanjing 210029, People's Republic of China.

ABSTRACT
JWA was recently demonstrated to be involved in cellular responses to environmental stress including oxidative stress. Although it was found that JWA protected cells from reactive oxygen species-induced DNA damage, upregulated base excision repair (BER) protein XRCC1 and downregulated PARP-1, the molecular mechanism of JWA in regulating the repair of DNA single-strand breaks (SSBs) is still unclear. Our present studies demonstrated that a reduction in JWA protein levels in cells resulted in a decrease of SSB repair capacity and hypersensitivity to DNA-damaging agents such as methyl methanesulfonate and hydrogen peroxide. JWA functioned as a repair protein by multi-interaction with XRCC1. On the one hand, JWA was translocated into the nucleus by the carrier protein XRCC1 and co-localized with XRCC1 foci after oxidative DNA damage. On the other hand, JWA via MAPK signaling pathway regulated nuclear factor E2F1, which further transcriptionally regulated XRCC1. In addition, JWA protected XRCC1 protein from ubiquitination and degradation by proteasome. These findings indicate that JWA may serve as a novel regulator of XRCC1 in the BER protein complex to facilitate the repair of DNA SSBs.

Show MeSH
Related in: MedlinePlus