Abstract ADP-ribosylation has long been recognised as a key regulator of essential signalling pathways, including the DNA damage response. However, only recent and ongoing technological advances are beginning to make it possible to investigate its distinct forms with molecular precision. Here, we design a ‘mono-ADP-ribosylation blocking’ strategy to develop sensitive, modular antibodies with high specificity for poly(ADP-ribosyl)ation. During peptide antigen generation, we identify a distinctive mass spectrometric signature that enables accurate mapping of poly(ADP-ribosyl)ation sites and helps prevent site mislocalization. Moreover, we affinity-mature mono-ADP-ribosylation and histone H3 site-specific antibodies. These tools reveal that, upon DNA damage, XRCC1 deficiency dramatically elevates the mono-ADP-ribosylation wave of PARP1 signalling, in addition to increasing poly(ADP-ribosyl)ation. This PARP1 hyperactivation leads to an increase in an unconventional form of ubiquitylation, recently shown to directly target mono-ADP-ribose in the DNA damage response and other signalling pathways. Consequently, XRCC1 loss enhances the recruitment of RNF114, the reader of this composite modification, to DNA lesions. These findings establish mono-ADP-ribosylation – and its ester-linked ubiquitylation – as key modifications induced by XRCC1 deficiency during DNA damage, revealed using tools we developed for precise and sensitive ADP-ribosylation detection.
Dauben et al. (2026) studied this question.