An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease. [Display omitted] •Phospho-guided strategy generates precisely ADP-ribosylated peptides•Recombinant site-specific and broad-specificity antibodies to ADPr were developed•PARP1 and histone H3 are predominantly mono-ADP-ribosylated upon DNA damage•Cellular levels of PARP1 and histone H3 mono-ADPr are modulated by PARG and ARH3 Site-specific and broad-specificity antibodies to ADP-ribosylation elucidate the prevalence of mono-ADP-ribosylation upon DNA damage.