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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
Published on: June 8, 2018
JWA regulates XRCC1 and functions as a novel base excision repair protein in oxidative-stress-induced DNA
Shouyu Wang1, Zhenghua Gong, Rui Chen
1Department 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.
Insights
JWA protein aids DNA repair by interacting with XRCC1, enhancing the base excision repair pathway. Reduced JWA impairs DNA single-strand break repair, increasing sensitivity to DNA damage.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- JWA protein involvement in cellular stress responses, including oxidative stress.
- Previous findings show JWA protects against reactive oxygen species-induced DNA damage.
- The precise mechanism of JWA in DNA single-strand break (SSB) repair remains elusive.
Purpose of the Study:
- To elucidate the molecular mechanism of JWA in regulating DNA single-strand break repair.
- To investigate the interaction between JWA and the base excision repair (BER) protein XRCC1.
- To determine JWA's role in cellular responses to DNA-damaging agents.
Main Methods:
- Cellular assays to assess SSB repair capacity and sensitivity to DNA-damaging agents (methyl methanesulfonate, hydrogen peroxide).
- Co-immunoprecipitation and immunofluorescence to study JWA-XRCC1 interaction and localization.
- Western blotting and proteasome inhibition assays to evaluate protein stability.
- Analysis of MAPK signaling pathway and E2F1 transcriptional regulation.
Main Results:
- Reduced JWA levels decreased SSB repair capacity and increased hypersensitivity to DNA damage.
- JWA interacts with XRCC1, translocates to the nucleus with XRCC1, and co-localizes with XRCC1 foci.
- JWA regulates nuclear factor E2F1 via the MAPK pathway, which transcriptionally controls XRCC1.
- JWA protects XRCC1 from ubiquitination and proteasomal degradation.
Conclusions:
- JWA plays a crucial role in DNA single-strand break repair.
- JWA functions as a novel regulator of XRCC1 within the base excision repair complex.
- JWA facilitates DNA SSB repair by stabilizing XRCC1 and modulating its expression.
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