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A Versatile Toolbox Reveals XRCC1 Deficiency–Driven Reshaping of the PARP1 Mono-ADP-Ribosylation Wave

ABSTRACT: ADP-ribosylation has long been recognized as a key regulator of essential signaling 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 designed a ‘mono-ADP-ribosylation blocking’ strategy to develop sensitive, modular antibodies with high specificity for poly-ADP-ribosylation. During peptide antigen generation, we identified a distinctive mass spectrometric signature that enabled accurate mapping of poly- ADP-ribosylation sites and helped prevent site mislocalization. Moreover, we affinity-matured 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 signaling, in addition to increasing poly-ADP-ribosylation. 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 signaling pathways. Consequently, XRCC1 loss enhanced the recruitment of RNF114, the reader of this composite modification, to DNA lesions. Our findings establish mono-ADP-ribosylation – and its downstream ubiquitylation – as a key modification triggered by XRCC1 deficiency during DNA damage, enabled by newly developed tools for precise and sensitive detection of ADP-ribosylation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ilian Atanassov

LAB HEAD: Ivan Matic

PROVIDER: PXD066208 | Pride | 2026-02-18

REPOSITORIES: Pride

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			Action	DRS
	Fig2A.txt	Txt
	Fig2BCD.txt	Txt
	SearchMonoDi_MaxQuant.zip	Other
	SearchMonoDi_MaxQuant_output.zip	Other
	SearchMonoOnly_MaxQuant.zip	Other

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