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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
SPRTN metalloprotease participates in repair of ROS-mediated DNA-protein crosslinks
Luke Erber1,2, Arnold S Groehler1, Cesar I Cyuzuzo1
1Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA.
Abstract:
Exposure to reactive oxygen species (ROS) can induce DNA-protein crosslinks (DPCs), unusually bulky DNA lesions that block replication and transcription and play a role in aging, cancer, cardiovascular disease, and neurodegenerative disorders. Repair of DPCs depends on the coordinated efforts of proteases and DNA repair enzymes to cleave the protein component of the lesion to smaller DNA-peptide crosslinks which can be processed by tyrosyl-DNA phosphodiesterases 1 and 2, nucleotide excision and homologous recombination repair pathways. DNA-dependent metalloprotease SPRTN plays a role in DPC repair, and SPRTN-deficient mice exhibit an accelerated aging phenotype and develop liver cancer early in life. We investigated the role of the SPRTN enzyme in the repair of DPCs produced by a free radical mechanism. Sprtn-deficient MEF cells treated with ionizing radiation had higher levels of total DPCs and exhibited greater sensitivity upon exposure to hydrogen peroxide and other crosslinking agents including cisplatin, phosphoramide mustard, and 1,2,3,4-diepoxybutane. Using a sensitive and accurate nanoLC-ESI+-MS/MS assay, we specifically measured the radical-induced crosslinking of thymidine in DNA crosslinking of thymidine in DNA to tyrosine in proteins (dT-Tyr) in the tissues of SPRTN hypomorphic (SprtnH/H) and wild type mice. Genomic DNA isolated from the tissues of SPRTN hypomorphic (SprtnH/H) mice exhibited higher levels of dT-Tyr in the liver, brain, heart, and kidney than wild-type animals. Overall, our results are consistent with the understanding that SPRTN has a role in maintaining genomic integrity upon exposure to ionizing radiation and endogenous reactive oxygen species.
Insights
The DNA repair enzyme SPRTN is crucial for fixing DNA-protein crosslinks (DPCs) caused by reactive oxygen species. SPRTN deficiency leads to increased DNA damage and sensitivity to various genotoxic agents, highlighting its role in preventing aging and cancer.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA-protein crosslinks (DPCs) are bulky DNA lesions induced by reactive oxygen species (ROS).
- DPCs impede DNA replication and transcription, contributing to aging and diseases like cancer.
- SPRTN, a DNA-dependent metalloprotease, is implicated in DPC repair, with its deficiency linked to accelerated aging and early liver cancer in mice.
Purpose of the Study:
- To investigate the role of the SPRTN enzyme in repairing DPCs generated by free radicals.
- To assess the impact of SPRTN deficiency on cellular sensitivity to various crosslinking agents.
- To quantify radical-induced DNA-protein crosslinks in vivo in SPRTN-deficient mice.
Main Methods:
- Utilized Sprtn-deficient mouse embryonic fibroblast (MEF) cells and SPRTN hypomorphic (SprtnH/H) mice.
- Treated cells with ionizing radiation, hydrogen peroxide, and chemical crosslinking agents.
- Employed a nanoLC-ESI+-MS/MS assay to measure thymidine-tyrosine (dT-Tyr) crosslinks in DNA and protein.
Main Results:
- Sprtn-deficient MEF cells showed elevated DPC levels and increased sensitivity to hydrogen peroxide and crosslinking agents.
- SPRTN hypomorphic mice exhibited significantly higher levels of dT-Tyr crosslinks in liver, brain, heart, and kidney tissues compared to wild-type mice.
- These findings confirm SPRTN's involvement in mitigating DNA damage from ROS and ionizing radiation.
Conclusions:
- SPRTN plays a vital role in the repair of DNA-protein crosslinks induced by reactive oxygen species and ionizing radiation.
- SPRTN deficiency compromises genomic integrity, leading to increased susceptibility to DNA damage.
- The study underscores SPRTN's importance in preventing age-related diseases and cancer by maintaining genomic stability.
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