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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 maintaining the stability of the XRCC1 protein. (A) JWA deficiency significantly enhanced the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the corresponding empty vector for 48 h, followed by exposure to cycloheximide (CHX) (50 μg/ml) for various time periods. Target proteins in whole-cell lysates were detected by immunoblotting using antibodies against XRCC1, LigIII and JWA. (B) The intensity of the XRCC1 and LigIII protein bands in (A) were analyzed by densitometry, after normalization to the corresponding β-actin level. The means ± SD are from three independent experiments. (C) The proteasome mediates the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, cells were incubated with or without of MG132 (10 μM) for 4 h, then the cells were cultured for another 30 min with or without 100 μM H2O2. Cell lysates were used for immunoblotting with antibodies against XRCC1, LigIII and JWA. β-Actin was used as a loading control. (D) An RFP-XRCC1 plasmid was transiently transfected into stable selected EGFP-C1 vector control or KD-JWA NIH-3T3 cells. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for 4 h. The cells were then fixed in methanol/acetone and counterstained with DAPI. The expression of RFP-XRCC1 in the cells (red) was observed under a fluorescent microscope. The nucleus of the cells was indicated by DAPI (blue). (E) NIH-3T3 or KD-JWA cells were transiently transfected with RFP-XRCC1 plasmid for 44 h, incubated with MG132 (10 μM) for 4 h, and western blotting was performed to confirm the levels of endogenous XRCC1 (lower molecule band) and exogenous XRCC1 (RFP-XRCC1). (F) Knocking down JWA results in the ubiquitylation and degradation of XRCC1. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for another 4°h. Cell lysates were used for IP with the XRCC1 antibody and then blotted for XRCC1, LigIII and ubiquitin. Western blotting for JWA and β-actin in whole-cell lysates was utilized to check the JWA knockdown efficiency and to ensure equal protein loading.
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Figure 6: JWA is required for maintaining the stability of the XRCC1 protein. (A) JWA deficiency significantly enhanced the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the corresponding empty vector for 48 h, followed by exposure to cycloheximide (CHX) (50 μg/ml) for various time periods. Target proteins in whole-cell lysates were detected by immunoblotting using antibodies against XRCC1, LigIII and JWA. (B) The intensity of the XRCC1 and LigIII protein bands in (A) were analyzed by densitometry, after normalization to the corresponding β-actin level. The means ± SD are from three independent experiments. (C) The proteasome mediates the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, cells were incubated with or without of MG132 (10 μM) for 4 h, then the cells were cultured for another 30 min with or without 100 μM H2O2. Cell lysates were used for immunoblotting with antibodies against XRCC1, LigIII and JWA. β-Actin was used as a loading control. (D) An RFP-XRCC1 plasmid was transiently transfected into stable selected EGFP-C1 vector control or KD-JWA NIH-3T3 cells. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for 4 h. The cells were then fixed in methanol/acetone and counterstained with DAPI. The expression of RFP-XRCC1 in the cells (red) was observed under a fluorescent microscope. The nucleus of the cells was indicated by DAPI (blue). (E) NIH-3T3 or KD-JWA cells were transiently transfected with RFP-XRCC1 plasmid for 44 h, incubated with MG132 (10 μM) for 4 h, and western blotting was performed to confirm the levels of endogenous XRCC1 (lower molecule band) and exogenous XRCC1 (RFP-XRCC1). (F) Knocking down JWA results in the ubiquitylation and degradation of XRCC1. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for another 4°h. Cell lysates were used for IP with the XRCC1 antibody and then blotted for XRCC1, LigIII and ubiquitin. Western blotting for JWA and β-actin in whole-cell lysates was utilized to check the JWA knockdown efficiency and to ensure equal protein loading.

Mentions: It was previously demonstrated that XRCC1 levels were reduced in stable JWA knockdown cells (42). We therefore postulated that JWA may be required for XRCC1 stability. When the cells were treated with CHX, an inhibitor of protein synthesis, we found that knockdown of JWA promoted the degradation of both endogenous XRCC1 and LigIII proteins in NIH-3T3 cells (Figure 6A and B). To determine whether the ubiquitin-proteasome pathway is responsible for XRCC1 degradation, cells were treated with the proteasome inhibitor MG132. We observed that loss of XRCC1 expression in JWA knockdown cells was inhibited by pretreatment with 10 μM MG132 for 4 h (Figure 6C). Similar effects were observed for LigIII (Figure 6C), suggesting that JWA inhibits the proteasomal degradation of these proteins. In order to confirm these data, an exogenous RFP-XRCC1 plasmid was transfected into stable selected vector control cells or KD-JWA cells with or without MG132 pretreatment. The transfected exogenous XRCC1 was also destabilized in JWA deficient cells (Figure 6D). The decreased expression of endogenous and exogenous XRCC1 in JWA knockdown cells was confirmed by western blotting (Figure 6E). However, the reduction in XRCC1 expression could be prevented by pretreatment with MG132 (Figure 6D and E).Figure 6.


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 maintaining the stability of the XRCC1 protein. (A) JWA deficiency significantly enhanced the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the corresponding empty vector for 48 h, followed by exposure to cycloheximide (CHX) (50 μg/ml) for various time periods. Target proteins in whole-cell lysates were detected by immunoblotting using antibodies against XRCC1, LigIII and JWA. (B) The intensity of the XRCC1 and LigIII protein bands in (A) were analyzed by densitometry, after normalization to the corresponding β-actin level. The means ± SD are from three independent experiments. (C) The proteasome mediates the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, cells were incubated with or without of MG132 (10 μM) for 4 h, then the cells were cultured for another 30 min with or without 100 μM H2O2. Cell lysates were used for immunoblotting with antibodies against XRCC1, LigIII and JWA. β-Actin was used as a loading control. (D) An RFP-XRCC1 plasmid was transiently transfected into stable selected EGFP-C1 vector control or KD-JWA NIH-3T3 cells. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for 4 h. The cells were then fixed in methanol/acetone and counterstained with DAPI. The expression of RFP-XRCC1 in the cells (red) was observed under a fluorescent microscope. The nucleus of the cells was indicated by DAPI (blue). (E) NIH-3T3 or KD-JWA cells were transiently transfected with RFP-XRCC1 plasmid for 44 h, incubated with MG132 (10 μM) for 4 h, and western blotting was performed to confirm the levels of endogenous XRCC1 (lower molecule band) and exogenous XRCC1 (RFP-XRCC1). (F) Knocking down JWA results in the ubiquitylation and degradation of XRCC1. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for another 4°h. Cell lysates were used for IP with the XRCC1 antibody and then blotted for XRCC1, LigIII and ubiquitin. Western blotting for JWA and β-actin in whole-cell lysates was utilized to check the JWA knockdown efficiency and to ensure equal protein loading.
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Figure 6: JWA is required for maintaining the stability of the XRCC1 protein. (A) JWA deficiency significantly enhanced the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the corresponding empty vector for 48 h, followed by exposure to cycloheximide (CHX) (50 μg/ml) for various time periods. Target proteins in whole-cell lysates were detected by immunoblotting using antibodies against XRCC1, LigIII and JWA. (B) The intensity of the XRCC1 and LigIII protein bands in (A) were analyzed by densitometry, after normalization to the corresponding β-actin level. The means ± SD are from three independent experiments. (C) The proteasome mediates the degradation of XRCC1 and LigIII. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, cells were incubated with or without of MG132 (10 μM) for 4 h, then the cells were cultured for another 30 min with or without 100 μM H2O2. Cell lysates were used for immunoblotting with antibodies against XRCC1, LigIII and JWA. β-Actin was used as a loading control. (D) An RFP-XRCC1 plasmid was transiently transfected into stable selected EGFP-C1 vector control or KD-JWA NIH-3T3 cells. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for 4 h. The cells were then fixed in methanol/acetone and counterstained with DAPI. The expression of RFP-XRCC1 in the cells (red) was observed under a fluorescent microscope. The nucleus of the cells was indicated by DAPI (blue). (E) NIH-3T3 or KD-JWA cells were transiently transfected with RFP-XRCC1 plasmid for 44 h, incubated with MG132 (10 μM) for 4 h, and western blotting was performed to confirm the levels of endogenous XRCC1 (lower molecule band) and exogenous XRCC1 (RFP-XRCC1). (F) Knocking down JWA results in the ubiquitylation and degradation of XRCC1. NIH-3T3 cells were transfected with JWA shRNA or the control vector. Forty-four hours later, the cells were incubated with or without of MG132 (10 μM) for another 4°h. Cell lysates were used for IP with the XRCC1 antibody and then blotted for XRCC1, LigIII and ubiquitin. Western blotting for JWA and β-actin in whole-cell lysates was utilized to check the JWA knockdown efficiency and to ensure equal protein loading.
Mentions: It was previously demonstrated that XRCC1 levels were reduced in stable JWA knockdown cells (42). We therefore postulated that JWA may be required for XRCC1 stability. When the cells were treated with CHX, an inhibitor of protein synthesis, we found that knockdown of JWA promoted the degradation of both endogenous XRCC1 and LigIII proteins in NIH-3T3 cells (Figure 6A and B). To determine whether the ubiquitin-proteasome pathway is responsible for XRCC1 degradation, cells were treated with the proteasome inhibitor MG132. We observed that loss of XRCC1 expression in JWA knockdown cells was inhibited by pretreatment with 10 μM MG132 for 4 h (Figure 6C). Similar effects were observed for LigIII (Figure 6C), suggesting that JWA inhibits the proteasomal degradation of these proteins. In order to confirm these data, an exogenous RFP-XRCC1 plasmid was transfected into stable selected vector control cells or KD-JWA cells with or without MG132 pretreatment. The transfected exogenous XRCC1 was also destabilized in JWA deficient cells (Figure 6D). The decreased expression of endogenous and exogenous XRCC1 in JWA knockdown cells was confirmed by western blotting (Figure 6E). However, the reduction in XRCC1 expression could be prevented by pretreatment with MG132 (Figure 6D and E).Figure 6.

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