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Updated: Jun 17, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
Biochemical evaluation of genotoxic biomarkers for 2-deoxyribonolactone-mediated cross-link formation with histones
Mi-Young Son1, Hyun-Ik Jun, Kwang-Geun Lee
1Department of Life Science, Dongguk University, Seoul, Korea.
Abstract:
Numerous environmental carcinogens involve radical formation interacting with DNA to produce 2-deoxyribonolactone (dL), a major type of oxidized abasic site, implicated in DNA strand breaks, mutagenesis, and formation of covalent DNA-protein cross-links (DPC). Studies showed major dL-specific DPC occurred due to reactions with DNA polymerase beta (Polbeta) dependent on native conformation, while other DPC formed involved nonenzymatic reactions of DNA binding proteins with dL lesions. Polbeta appeared to play a major role in alleviating the cytotoxic effects of neocarzinostatin, which was used as a dL-producing agent. When a duplex DNA containing a dL at a site-specific position was incubated with purified histones, DPC were formed between dL and each histone protein, including H1, H2A, H2B, H3, and H4. Comparative kinetic analysis of DPC formation with histones and Polbeta revealed two distinct mechanisms of dL-mediated DPC formation. The rate of DPC formation with Polbeta was approximately two orders of magnitude higher than that with various histone proteins. These results indicate that catalytic activity of Polbeta mediates rapid DPC formation between dL and this DNA repair enzyme, whereas nonenzymatic reactions of dL with histones form DPC more slowly. The abundance of histones and their constant interaction with DNA may nevertheless yield significant levels of DPC with dL, as biomarkers of dL-induced cytotoxicity. Overall, data suggest that occurrence of dL-mediated DPC with histones may contribute to the genotoxic effects of dL in DNA.
Insights
Environmental carcinogens create 2-deoxyribonolactone (dL) lesions in DNA, forming DNA-protein cross-links (DPCs). DNA polymerase beta (Polbeta) rapidly forms DPCs, while histones form them slowly, suggesting a role in genotoxicity.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Carcinogenesis
Background:
- Environmental carcinogens can induce DNA damage, specifically forming 2-deoxyribonolactone (dL), an oxidized abasic site.
- dL lesions are implicated in DNA strand breaks, mutagenesis, and the formation of covalent DNA-protein cross-links (DPCs).
- Previous studies highlighted the role of DNA polymerase beta (Polbeta) in forming dL-specific DPCs and its involvement in mitigating cytotoxic effects.
Purpose of the Study:
- To investigate the mechanisms and kinetics of DNA-protein cross-link (DPC) formation involving 2-deoxyribonolactone (dL) lesions.
- To compare DPC formation between dL and DNA polymerase beta (Polbeta) versus dL and histone proteins.
- To assess the potential contribution of dL-mediated DPC formation to the genotoxic effects of environmental carcinogens.
Main Methods:
- Incubation of duplex DNA containing site-specific dL lesions with purified histone proteins (H1, H2A, H2B, H3, H4).
- Incubation of duplex DNA containing site-specific dL lesions with DNA polymerase beta (Polbeta).
- Comparative kinetic analysis of DPC formation rates between dL and histones versus dL and Polbeta.
Main Results:
- DPCs were formed between dL lesions and all tested histone proteins (H1, H2A, H2B, H3, H4).
- DNA polymerase beta (Polbeta) formed dL-specific DPCs significantly faster than histone proteins, with rates approximately two orders of magnitude higher.
- Two distinct mechanisms were identified: rapid, catalytic DPC formation by Polbeta, and slower, nonenzymatic DPC formation by histones.
Conclusions:
- Catalytic activity of Polbeta mediates rapid DPC formation with dL lesions.
- Nonenzymatic reactions of dL with abundant histone proteins lead to slower DPC formation.
- dL-mediated DPC formation with histones may serve as biomarkers of dL-induced cytotoxicity and contribute to genotoxicity.
