Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA-protein crosslinks (DPCs) are harmful DNA lesions resulting from various endogenous and exogenous sources.
• Post-translational modifications (PTMs) such as ubiquitylation, SUMOylation, and ADP-ribosylation are early responses to DPCs, signaling repair pathways.
• Detecting PTM-conjugated DPCs is challenging due to their transient and low-abundance nature.

Purpose of the Study:

• To present a novel immunoassay for the purification and quantitative detection of PTM-conjugated DPCs in vivo.
• To enable the study of molecular mechanisms involved in repairing both enzymatic and non-enzymatic DPCs.
• To facilitate the discovery of small molecule inhibitors targeting DPC repair pathways.

Main Methods:

• The assay is based on the RADAR (rapid approach to DNA adduct recovery) assay, utilizing ethanol precipitation for DPC isolation.
• Genomic DNA containing DPCs undergoes normalization and nuclease digestion.
• Detection of ubiquitylated, SUMOylated, and ADP-ribosylated DPCs is achieved through immunoblotting with specific antibodies.

Main Results:

• The developed immunoassay successfully purifies and quantifies ubiquitylated, SUMOylated, and ADP-ribosylated DPCs.
• The assay can detect both drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs.
• This method provides a robust tool for investigating DPC repair.

Conclusions:

• The new immunoassay is effective for detecting and characterizing PTM-conjugated DPCs.
• This technique can advance the understanding of novel molecular mechanisms for DPC repair.
• The assay holds potential for discovering therapeutic agents targeting DPC repair pathways.