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Updated: Feb 27, 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
Mechanisms of DNA-protein crosslink repair
Julian Stingele1, Roberto Bellelli1, Simon J Boulton1
1The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Abstract:
Covalent DNA-protein crosslinks (DPCs, also known as protein adducts) of topoisomerases and other proteins with DNA are highly toxic DNA lesions. Of note, chemical agents that induce DPCs include widely used classes of chemotherapeutics. Their bulkiness blocks virtually every chromatin-based process and makes them intractable for repair by canonical repair pathways. Distinct DPC repair pathways employ unique points of attack and are crucial for the maintenance of genome stability. Tyrosyl-DNA phosphodiesterases (TDPs) directly hydrolyse the covalent linkage between protein and DNA. The MRE11-RAD50-NBS1 (MRN) nuclease complex targets the DNA component of DPCs, excising the fragment affected by the lesion, whereas proteases of the spartan (SPRTN)/weak suppressor of SMT3 protein 1 (Wss1) family target the protein component. Loss of these pathways renders cells sensitive to DPC-inducing chemotherapeutics, and DPC repair pathways are thus attractive targets for combination cancer therapy.
Insights
DNA-protein crosslinks (DPCs) are toxic lesions caused by chemotherapeutics. Distinct repair pathways, including TDPs, MRN complex, and SPRTN proteases, are crucial for genome stability and offer potential cancer therapy targets.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Covalent DNA-protein crosslinks (DPCs) are highly toxic DNA lesions formed by topoisomerases and other proteins.
- These lesions, induced by common chemotherapeutics, impede DNA repair and chromatin processes due to their bulky nature.
- Canonical repair pathways struggle to address DPCs, necessitating specialized repair mechanisms.
Purpose of the Study:
- To elucidate the distinct mechanisms and key players involved in repairing DNA-protein crosslinks (DPCs).
- To highlight the importance of DPC repair pathways in maintaining genome stability.
- To explore the therapeutic potential of targeting DPC repair pathways in cancer treatment.
Main Methods:
- Investigated the roles of Tyrosyl-DNA phosphodiesterases (TDPs) in directly hydrolyzing the protein-DNA linkage.
- Analyzed the MRE11-RAD50-NBS1 (MRN) nuclease complex's function in processing the DNA component of DPCs.
- Examined the activity of Spartan (SPRTN)/Wss1 family proteases in degrading the protein moiety of DPCs.
Main Results:
- TDPs directly cleave the covalent bond between protein and DNA in DPCs.
- The MRN complex excises DNA fragments containing DPCs.
- SPRTN/Wss1 proteases specifically target and remove the protein component of DPCs.
- Cells deficient in these DPC repair pathways exhibit sensitivity to DPC-inducing chemotherapeutics.
Conclusions:
- Distinct DPC repair pathways, utilizing TDPs, MRN, and SPRTN/Wss1, are essential for cellular survival and genome integrity.
- These pathways represent critical defense mechanisms against genotoxic chemotherapeutics.
- Targeting DPC repair pathways holds promise for developing novel combination cancer therapies.
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