Cell
Volume 183, Issue 4, 12 November 2020, Pages 1086-1102.e23
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An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation

https://doi.org/10.1016/j.cell.2020.09.055Get rights and content
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Highlights

  • 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

Summary

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.

Keywords

ADP-ribosylation
mono-ADP-ribosylation
MARylation
HFP1
PARP1
DNA damage
histones
chemical biology
antibodies

Cited by (0)

4

These authors contributed equally

5

Present address: Large Molecule Research, Pharma Research and Early Development, Roche Innovation Center Munich, 82377 Penzberg, Germany

6

Present address: Research Group of Mechanisms of DNA Repair, Max Planck Institute for Biology of Aging, 50931 Cologne, Germany

7

Lead Contact