Uncovering the DNA Adduct Interactome: Experimental Strategies to Identify Binding Proteins and Repair Pathways

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Abstract DNA is continuously exposed to endogenous and exogenous agents, including chemotherapeutic agents, environmental pollutants, and food-borne toxicants, resulting in covalent modifications of DNA bases known as DNA adducts. The biological impact of these adducts is primarily determined by their interaction with cellular proteins, which dictates whether the damage results in DNA repair, tolerance via translesion synthesis with potential mutagenesis, or cell death if the damage persists. Consequently, investigation of DNA adduct–protein interactions is essential for a deeper understanding of their biological processing. With this review, we provide a comprehensive overview of methodologies used to identify proteins that interact with covalent DNA modifications, with a focus on monovalent DNA adducts and a particular emphasis on proteins involved in DNA repair pathways. We introduce and critically discuss functional and biochemical assays used to assess repair pathways and isolate DNA adduct–binding proteins, as well as analytical strategies used to identify these proteins. We also provide a summary of approaches used for the further characterization of the DNA adduct–protein interactions and discuss how these insights can inform our understanding of the cellular impact of specific DNA adducts. Finally, we give an overview of the preparations necessary to perform the assays reviewed here. This work provides a roadmap to identify and characterize DNA adduct–binding proteins, establishing a foundation to elucidate their roles in DNA repair and cellular responses to damage.

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