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Updated: May 18, 2026
Quantification of γH2AX Foci in Response to Ionising Radiation
Published on: April 6, 2010
TOPORS modulates H2AX discriminating genotoxic stresses
Ki Moon Seong1, Seon Young Nam, Ji-Young Kim
1Division of Radiation Effect Research, Radiation Health Research Institute, Korea Hydro & Nuclear Power Co., Ltd., 132-703, Seoul, Korea. bovine@khnp.co.kr
Abstract:
H2AX plays an important role in chromatin reorganization implicated in DNA repair and apoptosis under various DNA damaging conditions. In this study, the interaction between TOPORS (topoisomerase I-binding protein) and H2AX was verified using mammalian cell extracts exposed to diverse DNA damaging stresses such as ionizing radiation, doxorubicin, camptothecin, and hydrogen peroxide. In vitro assays for ubiquitination revealed that TOPORS functions as a novel E3 ligase for H2AX ubiquitination. TOPORS was found to be dissociated from H2AX proteins when cells were exposed to oxidative stress, but not replication-inducing DNA damaging stress. The protein stability of H2AX was decreased when TOPORS was ectopically expressed in cells, and oxidative stresses such as hydrogen peroxide and ionizing radiation induced recovery of the H2AX protein level. Therefore, these biochemical data suggest that TOPORS plays a key role in the turnover of H2AX protein, discriminating the type of DNA damaging stress.
Insights
TOPORS acts as an E3 ligase for H2AX ubiquitination, influencing its protein stability. This interaction is stress-dependent, highlighting TOPORS
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Histone H2AX is crucial for DNA repair and apoptosis following DNA damage.
- Understanding the regulation of H2AX is vital for comprehending cellular responses to genotoxic stress.
Purpose of the Study:
- To investigate the interaction between TOPORS and H2AX under various DNA damaging conditions.
- To elucidate the role of TOPORS as an E3 ligase in H2AX ubiquitination and turnover.
Main Methods:
- Mammalian cell extracts were exposed to DNA damaging agents (ionizing radiation, doxorubicin, camptothecin, hydrogen peroxide).
- In vitro ubiquitination assays were performed to assess TOPORS' ligase activity.
- Protein-protein interactions and stability were analyzed under different stress conditions.
Main Results:
- TOPORS was confirmed to interact with H2AX and function as an E3 ligase for H2AX ubiquitination.
- TOPORS dissociated from H2AX under oxidative stress but not replication stress.
- Ectopic expression of TOPORS decreased H2AX stability, while oxidative stress recovered H2AX levels.
Conclusions:
- TOPORS plays a significant role in the turnover of H2AX protein.
- TOPORS's function in H2AX regulation is selective, differentiating between oxidative and replication-induced DNA damage.
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