Protein–Protein Cross-Linking by a DNA Damage-Derived Histone Modification

Nonenzymatic covalent modification (NECM) of lysine residues can be physiologically consequential. A NECM is formed by the oxidized abasic site (C4-AP), which is produced by DNA-damaging agents. C4-AP reacts with the ε-amine of histone lysines in nucleosome core particles (NCPs) to form an electrophilic 5-methylene pyrrolone NECM (K_MP). K_MP is also produced on histones in bleomycin-treated human cells. Here, we describe a molecule (1a) that yields K_MP by reacting directly with histones in NCPs. K_MP forms on lysines of all four core histones in the order H3 > H2A/H2B > H4. Biotinylated K_MP-containing NCPs prepared using 1a were incubated with HeLa nuclear lysates in the presence of glutathione. NCP-protein cross-links were observed by native PAGE. Protein-protein cross-links (PPCs) were enriched through intact NCP pull-down and identified via tryptic digests by LC-MS/MS. Model reactions demonstrate K_MP is more electrophilic than N-acyllysine post-translational modifications (PTMs) but does not form PPCs indiscriminately within NCPs. Enriched proteins are functionally biased, with overrepresentation of DNA binding, histone binding, histone PTMs, and transcription regulation. Proteins enriched by K_MP-containing NCPs produced by generating C4-AP on DNA were analyzed in parallel. Similar overrepresented functions were observed when C4-AP was introduced near the H3/H4 N-tails, whereas a distinct group of proteins was enriched when C4-AP was introduced near the H2A acidic patch. PPC formation by K_MP is modulated by the NCP environment. Combined with the known intracellular formation of K_MP, this study inspires investigating whether PPC formation by this NECM impacts cell function and viability.