A protein semisynthesis-based strategy to probe the impact of site-specific serine ADP-ribosylation on linker histone function

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study PARP biology. Here, we establish a generalizable strategy to prepare ADP-ribosylated peptides that are compatible with N-terminal, C-terminal and sequential protein ligation reactions. Two unique protein-assembly routes are employed to generate full-length linker histone constructs that are homogenously ADP-ribosylated at known DNA damage-dependent modification sites. We found that serine mono-ADP-ribosylation is sufficient to alleviate linker histone-dependent chromatin compaction, and that this effect is amplified by ADP-ribose chain elongation. Our work will greatly expand the scope of ADP-ribose-modified proteins that can be constructed via semisynthesis, which is rapidly emerging as a robust approach to elucidate the direct effects that site-specific serine mono- and poly-ADP-ribosylation have on protein function.