Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

We report here chemoenzymatic and fully synthetic methodologies to modify aspartate and glutamate side chains with ADP-ribose at specific sites on peptides. Structural analysis of aspartate and glutamate ADP-ribosylated peptides reveals nearquantitative migration of the side chain linkage from the anomeric carbon to the 2″-or 3″-ADP-ribose hydroxyl moieties. We find that this linkage migration pattern is unique to aspartate and glutamate ADP-ribosylation and propose that the observed isomer distribution profile is present in biochemical and cellular environments. After defining distinct stability properties of aspartate and glutamate ADP-ribosylation, we devise methods to install homogenous ADP-ribose chains at specific glutamate sites and assemble glutamate-modified peptides into full-length proteins. By implementing these technologies, we show that histone H2B E2 tri-ADP-ribosylation is able to stimulate the chromatin remodeler ALC1 with similar efficiency to histone serine ADP-ribosylation. Our work reveals fundamental principles of aspartate and glutamate ADP-ribosylation and enables new strategies to interrogate the biochemical consequences of this widespread protein modification.

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study poly(ADP-ribose) polymerase (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 homogeneously 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-ADPribosylation have on protein function.

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto full-length proteins with precise control over chain length and modification site. A series of semi-synthetic ADP-ribosylated histone proteins are prepared which demonstrate that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. Importantly, we found ALC1 selectively remodels "activated" substrates within heterogeneous nucleosome populations and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a method that is broadly applicable to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.