Abstract 1391 DNA-Protein Cross-Links: Formation and Repair Mechanisms

Genomic DNA wraps around histone octamers to form nucleosome core particles, the basic units of chromatin. Histones react with the apurinic/apyrimidinic site, one of the most abundant DNA lesions, to form reversible but long-lived DNA-protein cross-links (DPCs) at 3′-termini within single-strand breaks. These bulky 3′-histone-DPCs are deleterious and must be removed because the 3'-hydroxyl is required for gap-filling DNA repair synthesis. However, the chemical structure of 3′-histone-DPCs is not well characterized, and whether they are formed in cells and how they are repaired remain elusive. The obstacle is the lack of bioanalytic and chemical approaches to detect and synthesize 3′-histone-DPCs. In this work, we developed a sensitive liquid chromatography with tandem mass spectrometry workflow to characterize 3′-histone-DPCs, and demonstrated that histones cross-link to incised AP sites via Schiff bases. We also demonstrated for the first time the formation of 3′-histone-DPCs in human embryonic kidney 293T cells at unperturbed conditions. Moreover, we developed a chemical approach to synthesize stable and site-specific 3′-histone-DPCs. Our method employs oxime ligation to install an alkyne to 3′-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we found that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair. Overall, our work expands the fundamental understanding of DPC formation and repair mechanisms. This work is supported by the University of Texas at Austin.