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
(KMP). KMP is also produced on histones in bleomycin-treated
human cells. Here, we describe a molecule (1a) that yields
KMP by reacting directly with histones in NCPs. KMP forms on lysines of all four core histones in the order H3 >
H2A/H2B
> H4. Biotinylated KMP-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 KMP 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 KMP-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 KMP is modulated
by the NCP environment. Combined with the known intracellular formation
of KMP, this study inspires investigating whether PPC formation
by this NECM impacts cell function and viability.