Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics

Post-translational
modification of proteins with poly­(ADP-ribose)
(PAR) is an important component of the DNA damage response. Four PAR
synthesis inhibitors have recently been approved for the treatment
of breast, ovarian, and prostate cancers. Despite the clinical significance
of PAR, a molecular understanding of its function, including its binding
partners, remains incomplete. In this work, we synthesized a PAR photoaffinity
probe that captures and isolates endogenous PAR binders. Our method
identified dozens of known PAR-binding proteins and hundreds of novel
candidates involved in DNA repair, RNA processing, and metabolism.
PAR binding by eight candidates was confirmed using pull-down and/or
electrophoretic mobility shift assays. Using PAR probes of defined
lengths, we detected proteins that preferentially bind to 40-mer versus
8-mer PAR, indicating that polymer length may regulate the outcome
and timing of PAR signaling pathways. This investigation produces
the first census of PAR-binding proteins, provides a proteomics analysis
of length-selective PAR binding, and associates PAR binding with RNA
metabolism and the formation of biomolecular condensates.