Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodelling of PARP1 signalling

Domain: biology
Published: 2026-04-02
Editorial outcome: minor_revision

Abstract
ADP-ribosylation has long been recognized 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.

Introduction
ADP-ribosylation (ADPr) is a versatile post-translational modification (PTM) that regulates numerous cellular processes, including DNA repair, transcription, chromatin remodelling, signalling and cell death1-3. It involves the enzymatic transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD⁺) to target molecules, mediated primarily by members of the ADP-ribosyltransferase family2. This dynamic modification can occur as mono-ADPr, linear or branched poly-ADPr, and is reversed by ADP-ribosylhydrolases4. First identified in the 1960s5, ADPr has since emerged as a critical player in cellular stress responses. Its dysregulation is implicated in various diseases, including cancer and neurodegeneration, making it an attractive target for therapeutic intervention6.

The poly(ADP-ribose) polymerase (PARP) family of ADP-ribosyltransferases comprises 17 members in humans2. PARP1 is activated in response to DNA strand breaks, coordinating the recruitment of DNA repair machinery and chromatin relaxation7,8. As a central scaffolding protein in single-strand break repair (SSBR), XRCC1 is recruited to sites of DNA damage through its interaction with poly-ADPr synthesized by PARP1/PARP29, enabling the formation of repair complexes. Loss of XRCC1 has been shown to cause elevated poly-ADPr levels through PARP1 hyperactivation, which has been linked to neurodegeneration10,11. By restraining PARP1 recruitment and catalytic activity during base excision repair (BER), XRCC1 prevents persistent PARP1 binding to BER intermediates. Recent work has further clarified how XRCC1 loss influences the temporal dynamics of PARP1 signalling. In particular, Demin et al.10 demonstrated that in MMS-treated XRCC1-deficient cells, early PARP1 hyperactivation drives rapid NAD+ consumption, suppressing PARP1 auto-modification at later time points. These findings indicate that XRCC1 not only restrains PARP1 activation but also preserves NAD+ availability necessary for sustained ADPr responses. Moreover, sustained PARP1 activity induced by XRCC1 deficiency has been shown to inhibit transcriptional recovery after DNA damage12.

ADPr is difficult to study due to its multi-layered complexity. Its biological complexity stems from the diversity of its enzymes, substrates, and cellular contexts involved, with conjugation chemistries ranging from O-glycosidic (serine and tyrosine), S-glycosidic (cysteine), N-glycosidic (arginine) to ester linkages on aspartate and glutamate. Some conjugation chemistries have remained elusive for decades. A prime example of this is serine ADPr, discovered less than ten years ago despite its abundance13-15. The structural complexity of ADPr arises from its versatility to form both mono-ADPr and polymeric chains. This biological, chemical and structural complexity has historically hindered the study of ADPr by limiting the development of tools. The past decade has seen a rapid expansion of methodologies and tools that have advanced the study of ADPr16, but a comprehensive toolbox for ADPr remains in development.

Our laboratory has contributed to extending the ADPr toolkit by converting our discoveries of serine ADPr by HPF1/PARP114,15 into a programmable technology for generating ADPr peptides and, subsequently, anti-ADPr antibodies and ADP-ribosylated nucleosomes17-21. This includes the first site-specific ADPr antibodies21, antibodies for mono-ADPr detection in diverse signalling pathways17,18,21-30 and in the context of ADP-ribosyl-ubiquitylation31-36. Serine ADPr technology is crucial because it not only generates antigens for phage display-based clone selection, but also enables in-depth validation and antibody characterization. For example, the resistance of serine ADPr to hydroxylamine recently demonstrated the specificity of a reagent we developed for detecting ADP-ribosyl-ubiquitylation33.

Phage display rapidly identifies multiple antigen binders in non-standard formats, but converting them to immunoglobulins through cloning is impractical and time-consuming. Fortunately, the SpyTag/SpyCatcher37 protein ligation technology now rapidly produces various antibody formats, including rabbit, mouse and human synthetic immunoglobulins, along with site-specific labeling with horseradish peroxidase (HRP), biotin, and other tags38. Immunoblotting with bivalent antibodies carrying three HRP molecules offers a more sensitive, simpler and faster alternative to standard protocols based on secondary antibodies38, greatly improving ADPr detection17,18. Beyond synthetic antibodies, we recently applied the SpyTag system to engineer a protein domain for enriching and detecting cellular ADP-ribosyl-ubiquitylation33.

While PARP1 has long been associated exclusively with poly-ADPr, our antibodies revealed that mono-ADPr is in fact more abundant during the DNA damage response21, illustrating the power of new tools to drive unexpected discoveries. This observation is explained by the rapid turnover of poly-ADPr, which constitutes an initial wave of PARP1 signalling, followed by a slower but more persistent accumulation of mono-ADPr18. This second signalling wave recruits RNF114, DTX3L and other proteins to DNA lesions18. RNF114, a ubiquitin E3 ligase that binds both mono-ADPr18 and ubiquitin, serves as a reader of ADP-ribosyl-ubiquitylation (ADPrUb), an emerging composite PTM31-34,39 we recently showed targets specific serine mono-ADPr marks on histones and PARP1 upon DNA damage33.

Here, we extend our serine ADPr technology20,21 to develop SpyTag-based antibodies for sensitive and specific poly-ADPr detection across multiple applications. Employing diverse serine ADPr substrates, we characterize and validate our poly-ADPr antibodies alongside affinity-matured mono-ADPr and H3S10/S28ADPr antibodies. Applying this toolset, we find that PARP1 hyperactivation resulting from XRCC1 deficiency increases not only poly-ADPr, as previously reported10,11, but also mono-ADPr. Additionally, our SpyTag ZUD reagent33 reveals that XRCC1 loss elevates cellular levels of ADPrUb, enhancing RNF114 recruitment to sites of DNA damage.

Results
A ‘mono-ADP-ribosylation blocking’ strategy
Previously, we established a programmable PARP1/HPF1 reaction for the rapid and scalable generation of serine mono-ADPr peptides, which enabled the development, validation and characterization of site-specific, mono-ADPr and pan-ADPr antibodies18,20,21. Here, we aimed to adapt our strategy to generate modular antibodies specific for poly-ADPr. Inspired by the possibility of extending the initial ADP-ribose attached to serine40, we employed a simple protocol that does not require separation of different ADP-ribosylated species. Specifically, following the serine mono-ADPr reaction, we purified the mono-ADPr peptide from the reaction components, as in our original approach21, and subsequently added PARP1, but not HPF1, to extend the mono-ADP-ribose with additional ADP-ribose units (Fig. 1A and Supplementary Fig. 1A). While we generated almost purely mono-ADPr peptides21 (Fig. 1B and Supplementary Fig. 1B,C), we were unable to further ADP-ribosylate more than half of the initial mono-ADPr peptide (Fig. 1C and Supplementary Fig. 1B,D). Therefore, we redirected our strategy from peptides to automodification of full-length PARP1, which results in its complete serine poly-ADPr21. However, this approach did not yield any antibodies, possibly due to interference from the highly abundant and long poly-ADP-ribose chains on PARP1 during its immobilization for phage display. To circumvent these challenges, we returned to poly-ADPr peptides and drew inspiration from our successful guided selection strategy, in which the unmodified peptide steered the selection towards antibodies that recognize the ADP-ribose moiety rather than other regions of the target peptide21. The obvious extension of this strategy to poly-ADPr would involve using a poly-ADPr-modified peptide as the antigen and an unmodified counterpart peptide for blocking. However, this approach would require a pure poly-ADPr peptide, which we were unable to generate, so we reasoned that blocking with a mono-ADPr-modified peptide would prevent the selection of antibodies that recognize the abundant contaminating mono-ADPr species (Fig. 1A and Supplementary Fig. 1A). With this strategy, our ability to generate only partially poly-ADP-ribosylated peptides (Fig. 1C and Supplementary Fig. 1B) would be sufficient for the selection of antibodies specific for poly-ADPr.

Given the value of the SpyTag system in generating modular antibodies18, we chose to implement it for our poly-ADPr antibodies. As illustrated in the scheme (Supplementary Fig. 1E,F), the monovalent antibody can be rapidly and reliably converted into a bivalent format using various catchers for coupling38, such as an HRP-coupled antibody for immunoblotting or a synthetic IgG antibody for immunofluorescence18,38.

Diagnostic ions for identification of di-ADP-ribosylation sites
During our mass spectrometric analysis, we observed fragment ions that appeared exclusively in spectra of poly-ADPr peptides (Supplementary Fig. 1C,D). To test the hypothesis that these peaks represent specific diagnostic ions for poly-ADPr, we modified a second peptide and observed the same fragmentation pattern for its di- and tri-ADPr forms, but not for the mono-ADPr form (Fig. 2A). From the structure of di-ADP-ribose, we interpreted these ions as corresponding to singly charged AMP+phosphoribose, ADP+phosphoribose, ADPr+AMP and di-ADPr (Fig. 2A and Supplementary Fig. 2A,B). These additional ions are specific for poly-ADPr because their formation requires two covalently attached ADP-ribose moieties. In contrast, mono-ADPr diagnostic ions can originate either from the terminal ADP-ribose within a poly-ADPr chain or from mono-ADPr.

Given the importance of specific diagnostic ions in our recent approach to distinguish ADP-ribosyl-ubiquitylation from the co-occurrence of ADPr and ubiquitylation as separate modifications33, we reasoned that poly-ADPr-specific diagnostic ions could likewise serve as an effective means to distinguish poly- from mono-ADPr. Therefore, building on our reanalysis strategy41, we searched for di-ADP-ribosylated peptides in published EThcD data42. After manual validation of MS/MS spectra, we confidently identified di-ADPr on primary serine ADPr targets14,15,42, including histones and PARP1 (Fig. 2B,C; Supplementary Fig. 2C and Supplementary Data 1).

Given the risk of site mislocalization in ADPr proteomics14,43, we hypothesized that di-ADPr could contribute to this issue, as computational proteomics analyses typically consider only mono-ADPr. We reasoned that if search engines are not allowed to consider di-ADPr as a potential modification, they may be forced to assign two separate mono-ADPr modifications to peptides actually modified by a single di-ADPr. Consequently, a di-ADPr-modified residue could be misassigned as mono-ADP-ribosylated, with an adjacent residue incorrectly annotated as the second mono-ADPr site. To test whether interpreting di-ADPr as two mono-ADPr modifications causes such mislocalization, we compared two reanalyses: one considering only mono-ADPr and another allowing both mono- and di-ADPr as variable modifications. We observed that restricting the search to mono-ADPr can result in misassignment, with ADPr incorrectly localized to a lysine instead of the adjacent serine modified by di-ADPr (Fig. 2D). In the search allowing for di-ADPr, the detection of poly-ADPr-specific diagnostic ions, along with additional fragment ions between the lysine and serine – unannotated after the mono-ADPr-only search – demonstrates that only the serine residue is modified by di-ADPr.

Overall, the diagnostic ions identified from peptide antigens designed for poly-ADPr antibody generation enabled mapping of di-ADPr sites in proteomics analyses, correcting mislocalizations inherent to standard workflows that consider only mono-ADPr.

Modular Antibodies for versatile and sensitive detection of Poly-ADPr
To generate modular antibodies specific for poly-ADPr we employed a mixture of mono- and poly-ADP-ribosylated histone H3S10 peptides (Fig. 1C and Supplementary Fig. 1B). Building on our previous phage display approach21, we introduced a key modification: guided selection toward poly-ADPr through our mono-ADPr blocking strategy (Fig. 1A and Supplementary Fig. 1A).

We first assessed the specificity of two selected poly-ADPr antibodies (AbD64138, AbD64235) by taking advantage of a feature of serine ADPr: the inability of PARG to cleave the bond between serine and the initial ADP-ribose21, despite its ability to hydrolyse tyrosine and aspartate/glutamate ADPr17,44. Specifically, we automodified PARP1 in the presence of HPF1, resulting in nearly complete poly-ADPr of PARP1. Addition of PARG to the reaction completely reduced poly-ADP-ribosylated PARP1 to mono-ADP-ribosylated PARP121, resulting in mono- and poly-ADPr standards for antibody validation. While our mono-ADPr antibody18 recognized only mono-ADP-ribosylated PARP1, our poly-ADPr antibodies showed no reactivity toward mono-ADPr but produced a strong signal for poly-ADP-ribosylated PARP1 (Fig. 3A and Supplementary Fig. 3A). As expected, the 10H antibody and the WWE-based reagent45 proved specific for poly-ADPr, although 10H detected only long ADPr polymers (Fig. 3A). Similar to the WWE-based reagent, our antibodies detected the full spectrum of poly-ADPr chain lengths in immunoblotting. To ensure a fair comparison of signal intensities, we tested the reagents at two different dilutions, starting with those recommended for each tool. However, using our antibodies at the same concentration (1 µg/ml) immediately caused signal bleaching. Even after reducing the antibody concentration tenfold, the sensitivity of our antibodies remained so high that oversaturation occurred after just one second of exposure. To enable a more accurate comparison without signal saturation, we performed a dot blot using a 1:2 dilution series of poly-ADP-ribosylated PARP1 (Fig. 3B and Supplementary Fig. 3B,C). Antibodies were tested at either the recommended concentrations, or at a common dilution of 0.1 µg/ml. In the HRP-SpyTag format, our antibodies produced significantly stronger signal intensities than both the WWE-based reagent and the CST pan-ADPr antibody, while the 10H antibody failed to generate any detectable signal at this dilution.

Our antibodies are not restricted to serine-ADPr but also detect poly-ADPr on asparate and glutamate, indicating that they recognise poly-ADPr irrespective of the underlying amino acid (Fig. 3C and Supplementary Fig. 3D). By establishing an optimized electrophoretic separation method for resolving oligo-ADP-ribosylated peptides, we found that the antibodies recognize poly-ADPr chains as short as di-ADPr (Supplementary Fig. 3E). Moreover, they efficiently detect free poly-ADP-ribose (Supplementary Fig. 3F). Together, these results illustrate the broad substrate recognition of our poly-ADPr antibodies.

Next, we assessed the performance of these tools in cells treated with H₂O₂ for ten minutes, with or without the PARP inhibitor Olaparib (Fig. 3D and Supplementary Fig. 3G). All reagents were used at their optimal dilutions. No nonspecific binding was detected, as evidenced by the absence of signal in Olaparib-treated samples (lanes 2 and 4 of each blot). Our antibodies exhibited high sensitivity, even when compared to commercially available reagents used at tenfold higher concentrations. Importantly, even without PARG inhibition previously required for detection46, this exceptional sensitivity enables detection of poly-ADPr in unperturbed cells (Fig. 3E and Supplementary Fig. 3H), where its generation has been linked to unligated Okazaki fragments46. This sensitivity is particularly critical when basal ADPr levels are low, such as RPE1 cells grown under hypoxia. Even under these challenging conditions, our antibodies detected PARP1-dependent poly-ADPr, whereas commercial reagents failed to produce a detectable signal (Fig. 3E and Supplementary Fig. 3H).

To further assess specificity, we performed immunofluorescence microscopy using the synthetic IgG format. We tailored dilutions and microscope settings to maximize the performance of each reagent. While signal intensities are not directly comparable, our antibodies provided a better signal-to-noise ratio in untreated versus H₂O₂-treated cells (Fig. 3F and Supplementary Fig. 4A) and performed best with methanol fixation (Supplementary Fig. 4B). Given the strong influence of antibody dilution on background signal, we tested multiple dilutions, all of which maintained poly-ADPr specificity with minimal nonspecific binding (Supplementary Fig. 4C). Next, inspired by the simultaneous detection of mono- and poly-ADPr using ADP-ribose binding reagents47, we leveraged the format switching enabled by SpyTag technology to detect these ADPr forms concurrently in cells. Specifically, we coupled the poly-ADPr antibody with a mouse Fc SpyCatcher and the mono-ADPr antibody with a rabbit Fc SpyCatcher. To prevent channel bleed-through, we used two fluorophores with distinct emission peaks and employed line sequencing during imaging. The results demonstrate that, following a ten-minute H₂O₂ treatment, both mono- and poly-ADPr can be detected simultaneously in the same cells (Supplementary Fig. 4D). Poly-ADPr staining appeared as nuclear foci, suggesting the localized activation of PARP enzymes at specific chromatin sites, where polymeric ADP-ribose accumulates. In contrast, mono-ADPr exhibited a more diffuse nuclear distribution, consistent with its broader roles across chromatin.

Affinity-matured mono-ADPr and H3S10/S28ADPr antibodies
Whereas our poly-ADPr antibodies are both sensitive and specific as original clones (Fig. 3), our other antibodies have shown specificity but limited sensitivity21. Although the original clones of these antibodies have advanced studies across multiple laboratories22,23,28,30,35,48, the affinity maturation of AbD332042R1 into AbD4364718 demonstrates how increased sensitivity can broaden their applicability17,23,24,27,36. Therefore, to increase the sensitivity of our site-specific H3S10/S28ADPr antibody AbD33644 and our mono-ADPr antibody AbD3320521, we applied the affinity maturation strategy (Fig. 4A) that yielded AbD4364718. While both AbD33205 and AbD33204/AbD43647 are specific for mono-ADPr, they exhibit differences in substrate preference18,21, making them complementary and valuable for different applications17,22,23,27,28,30,35,36,48. For example, only AbD33204/AbD43647, but not AbD33205, recognize free ADP-ribose18,21.

We performed ELISA analyses on the selected antibodies – the new mono-ADPr clone AbD41122 and the new H3S10/S28 clones AbD55568 and AbD55558 – using PARP1S507, H3S10, H3S28, and H1.4S149 mono-ADPr peptides21, unmodified H3(1-21) peptide and free mono-ADP-ribose (Fig. 4B). As with our previously developed antibodies18,21, none of the selected clones bound the unmodified peptide. Like its parental clone21, AbD41122 did not recognize free mono-ADP-ribose and showed distinct context dependencies (Fig. 4B). AbD41122 and AbD43647 differ substantially in substrate preference, and no single clone is sufficient to capture the full diversity of mono-ADPr (Supplementary Fig. 5A). Moreover, AbD41122 robustly detects starvation-induced mono-ADPr on RNA49, while AbD43647 does so only weakly and with nonspecific background (Fig. 4C and Supplementary Fig. 5C), making AbD41122 the preferred reagent for specific detection of mono-ADPr on RNA. The two H3S10/S28 clones retained the specificity of their parental clone AbD3364421, primarily recognizing the H3S10ADPr site and, to a lesser extent, the less abundant H3S28ADPr site (Fig. 4B and Supplementary Fig. 5B), consistent with the sequence similarities surrounding these two sites.

Next, we used AbD41122 for immunoblotting and immunofluorescence of cells treated with DNA-damaging agents and the PARP inhibitor Olaparib. This new antibody was more sensitive than its parental clone AbD3320521 and comparable to AbD4364718, although it recognized a different pattern (Fig. 4D,E and Supplementary Fig. 5D,E). The new H3S10/S28ADPr clones were more sensitive than their parental clone but exhibited similar background signal, which was largely eliminated by sulfuric acid histone extraction (Fig. 4F and Supplementary Fig. 5F-H).

Next, we treated cells with a PARG inhibitor, which massively increases cellular poly-ADPr levels, as previously used to demonstrate the mono-ADPr specificity of antibodies18. While PARG inhibition led to a strong increase in signal detected by both pan- and poly-ADPr antibodies, the AbD41122 signal did not increase, confirming its mono-ADPr specificity (Fig. 4G and Supplementary Fig. 5I). The distinct staining pattern in response to PARG inhibition compared to AbD43647 further illustrates the complementarity of these antibodies. For the site-specific antibodies, we observed a smear above the main H3 band, suggesting that these clones recognize both mono- and poly-ADPr on H3S10/S28 (Fig. 4G).

The ability to convert these modular antibodies into bivalent biotin-coupled reagents via SpyTag/SpyCatcher ligation – offering improved pull-down efficiency compared to IgG (Supplementary Fig. 5J) – makes AbD41122 suitable for mono-ADPr enrichment (Supplementary Fig. 5K). Combining the biotin SpyTag format for pulldown with the HRP SpyTag format for immunoblotting can enhance signal detection and avoid antibody leakage18,33.

XRCC1 deficiency elevates the mono-ADPr wave of PARP1 signalling
Having developed modular antibodies for the specific and sensitive detection of mono-ADPr and poly-ADPr in cellular contexts (Figs. 3 and 4; Supplementary Fig. 5L), we proceeded to explore the different forms of ADPr generated by PARP1 following its hyperactivation induced by XRCC1 deficiency10,11. Our discovery of mono-ADPr as a second wave of PARP1 signalling18, together with our tools, allows us to move technically and conceptually beyond the detection of pan-ADPr signals, which following DNA damage have often been assumed to represent poly-ADPr, including in the context of XRCC1 deficiency11. Given that ARH3 deficiency – which, like XRCC1 mutation, causes neurodegeneration – massively increases cellular mono-ADPr levels21,30,50, we were intrigued to test whether XRCC1 deficiency affects mono-ADPr.

Consistent with a report that XRCC1 prevents PARP1 trapping10, we and others observed an enhanced accumulation of endogenous PARP1 at DNA lesions in XRCC1 KO cells compared to WT (Supplementary Fig. 6A)51. As the effect of XRCC1 deficiency on ADPr was previously studied after recovery from H₂O₂-induced DNA damage11, we first examined mono- and poly-ADPr under these conditions. As anticipated11, poly-ADPr levels, particularly on histones, increased in DNA-damaged XRCC1-deficient cells compared to WT (Fig. 5A and Supplementary Fig. 6B). Additionally, we observed a marked increase in mono-ADPr, with the two antibodies detecting distinct bands, as expected from their complementary specificities (Fig. 4). We observed higher mono- and poly-ADPr levels in WT compared to XRCC1 KO cells after recovery from methyl methanesulfonate (MMS) treatment (Supplementary Fig. 6C). This late decrease in PARP1 activity in XRCC1-deficient cells is consistent with the model in which early PARP1 hyperactivation consumes NAD+, thereby limiting auto-modification and polymer extension at later time points10. 

Next, as previously done for ARH3 deficiency21, we investigated ADPr during the active phase of DNA damage – a condition under which a previous study using a pan-ADPr antibody did not detect differences between XRCC1 KO and WT cells11. Building on our prior evidence highlighting the importance of timing in ADPr pattern dynamics following DNA damage17,18,21, we applied continuous H₂O₂ and MMS exposure and observed that the absence of XRCC1 elevates not only poly- but also mono-ADPr even during ongoing DNA damage (Fig. 5B and Supplementary Fig. 6D, E). However, after 90 minutes of MMS-induced DNA damage, WT cells showed higher levels of poly-ADPr and core histone mono-ADPr than XRCC1 KO cells (Supplementary Fig. 6E), in line with reduced PARP1 activity at later stages of damage in XRCC1-deficient cells10.

Both forms of ADPr are generated by PARP1, as their signals were largely abolished by PARP1 inhibition using either Olaparib or the PARP1-specific inhibitor AZD9574 (Fig. 5C), yet persisted after boiling (Supplementary Fig. 6F), suggesting modification on serine residues17. We were intrigued by a ~25–30 kDa band that was strongly increased in XRCC1 KO cells following both H₂O₂ and MMS treatments, and was more prominently detected by AbD41122 than by AbD43647 (Fig. 5A,B and Supplementary Fig. 6B,E). This band is interesting since mono-ADPr on this target is not a remnant of poly-ADPr, given that it is unaffected by PARG inhibition18. Through perchloric acid enrichment, which solubilizes histone H1 while leaving most other proteins insoluble (Supplementary Fig. 6G), we identified this mono-ADPr–exclusive target as histone H1 (Fig. 5D). This identification was further confirmed by mutating the main ADPr sites on H115,42 (Fig. 5E). The sustained mono-ADPr signal on H1 in XRCC1-deficient cells is intriguing, as studies indicate that mono-ADPr weakens H1–DNA interactions and can promote its displacement from chromatin during DNA damage52, a process that may contribute to chromatin relaxation at damage sites. Prolonged H1 mono-ADPr may therefore reflect extended chromatin remodeling resulting from persistent repair intermediates.

Next, we investigated the behavior of mono- and poly-ADPr in response to XRCC1 deficiency and various genotoxic treatments. Exploring ADPr dynamics is essential to accurately define how distinct forms of ADPr respond to different treatments and genetic perturbations, as these can influence not only the extent but also the timing of ADPr induction, as we have shown previously17,18,21. We first applied our live-cell imaging approach to monitor the recruitment of mono- and poly-ADPr probes to sites of DNA damage18. XRCC1 deficiency led to a substantial rise in poly-ADPr and mono-ADPr, especially at later time points (Fig. 6A), consistent with our immunoblotting results (Fig. 5).

To further dissect the dynamics of poly- and mono-ADPr, we treated XRCC1 KO and WT cells with distinct types of DNA damage – H₂O₂, MMS and camptothecin (CPT) – across varying durations. Intriguingly, XRCC1 deficiency alters ADPr dynamics in a damage type–specific manner (Fig. 6B,C). CPT primarily induced poly-ADPr, with mono-ADPr detectable only in XRCC1-deficient cells. H₂O₂ led to an overall elevation of both mono- and poly-ADPr, whereas MMS induced a much more rapid ADPr response in XRCC1-deficient cells compared to WT (Fig. 6B,C and Supplementary Fig. 7A,B). We observed a similar ADPr pattern in XRCC1-deficient patient fibroblasts (Supplementary Fig. 7C). Overall, mono-ADPr appeared more stable and homogeneously distributed among the cell population, while poly-ADPr remained transient and spatially heterogeneous – particularly after H₂O₂ treatment (Fig. 6B). This more heterogeneous distribution of poly-ADPr likely reflects its transient nature, whereby rapid turnover renders its detection highly sensitive to subtle temporal or cell-to-cell differences in PARP1 activity.

Immunoblotting further revealed treatment-specific temporal dynamics: H₂O₂ induced distinct sequential waves of mono- and poly-ADPr; with MMS, the waves were temporally closer; and with CPT, they largely overlapped, resulting in low-levels of mono-ADPr detectable only under conditions of PARP1 hyperactivation caused by XRCC1 loss (Fig. 6C,D). To contextualize XRCC1 deficiency, we compared it with ARH3 loss, as both are linked to neurodegeneration and share elevated mono-ADPr (Figs. 5,6)21,30,50. While both conditions lead to increased mono-ADPr, ARH3 deficiency affects exclusively mono-ADPr. In contrast, XRCC1 loss increases both mono- and poly-ADPr, displaying a distinct pattern compared to ARH3-deficient cells (Supplementary Fig. 7D), consistent with elevated mono-ADPr arising from altered PARP1 activation rather than impaired mono-ADPr removal.

Collectively, these findings demonstrate the profound impact of XRCC1 deficiency not only on the magnitude but also on the timing of ADPr (Fig. 6D). Importantly, this includes mono-ADPr – the long-overlooked second wave of PARP1 activity18 – which we can now directly visualize (Fig. 4). Crucially, the increase in both mono- and poly-ADPr caused by XRCC1 loss allowed us to apply our tools to uncover how different genotoxic agents distinctly affect these two signalling waves.

Altered mono-ADPr dynamics in XRCC1-deficient cells involve PARG-dependent turnover
Having observed that mono-ADPr levels increase in the absence of XRCC1 (Figs. 5, 6) we next investigated whether this signal arises from altered PARG-dependent turnover of poly-ADPr. To address this, we examined the effect of PARG inhibition (PARGi) on the accumulation of serine mono-ADPr in XRCC1 KO cells. Although PARG can remove mono-ADPr from tyrosine and aspartate/glutamate17,44, it cannot hydrolyze serine-linked mono-ADPr21. PARG inhibition therefore provides a means to assess how much serine mono-ADPr arises from poly-ADPr degradation. Consistent with our previous studies18,21, when PARG inhibition is applied throughout genotoxic treatment, mono-ADPr is largely PARG-dependent, as evidenced by the strong reduction in mono-ADPr signal (Fig. 7A and Supplementary Fig. 7E). Notably, the extent of this reduction is similar in WT and XRCC1 KO cells, consistent with elevated mono-ADPr levels in XRCC1 KO cells arising from increased levels of the PARG substrate poly-ADPr rather than from increased PARG activity.

Next, we extended the late-stage PARG inhibition approach of Demin et al.10 by applying short PARGi pulses (5, 10, or 15 min before harvest) during continuous 45 min H₂O₂ treatment, the condition showing the largest XRCC1-dependent increase in mono-ADPr relative to WT cells. This strategy provides a direct readout of acute PARG-dependent conversion of poly-ADPr to mono-ADPr, allowing us to determine whether persistent mono-ADPr observed at late time points in XRCC1 KO cells is generated by rapid hydrolysis of pre-formed poly-ADPr. Consistent with low poly-ADPr levels at this late stage of ADPr signalling, short pulses of PARG inhibition had only a mild effect on mono-ADPr (Fig 7B).

Together, our results provide no evidence that XRCC1 deficiency measurably alters PARG activity. Instead, they indicate that mono-ADPr accumulation in XRCC1-deficient cells reflects a time-dependent contribution of PARG-mediated turnover, with strong dependence on PARG activity at early stages revealed by full PARG inhibition (Fig. 7A), but only limited sensitivity to late-stage PARG inhibition (Fig. 7B).

XRCC1 deficiency increases RNF114 accumulation at sites of DNA damage and elevates cellular levels of DNA damage-induced ADP-ribosyl-ubiquitylation.
Our finding that XRCC1 deficiency leads to elevated levels of DNA damage-induced mono-ADPr, prompted us to investigate the downstream consequences of this increase. Specifically, we tested the hypothesis that XRCC1 loss enhances recruitment of the ubiquitin E3 ligase RNF114 to sites of DNA damage, based on our finding that RNF114 recruitment increases when mono-ADPr levels are elevated18. Using live-cell imaging to track GFP-tagged RNF11418, we observed increased accumulation at DNA lesions in XRCC1-deficient cells compared to WT cells (Fig. 8A). Given that RNF114 binds not only mono-ADPr18 but also ubiquitin, functioning as a reader of ADPrUb31-34, we reasoned that XRCC1 deficiency might lead to increased cellular levels of this composite modification. In this context, we recently demonstrated that DNA damage-induced serine mono-ADPr marks on histones and PARP1 are further modified by this unconventional ester-linked ubiquitylation33. We were particularly encouraged by our observation that ADPrUb on serine is increased in cells lacking ARH333, a condition that leads to a marked accumulation of mono-ADPr21,30,50 (Supplementary Fig. 7D).

To detect cellular ADPrUb, we employed our recently developed method combining biotin-coupled SpyTag-ZUD pulldown followed by immunoblotting with mono-ADPr antibodies and HRP-coupled SpyTag-ZUD33. While our mono-ADPr antibodies have been widely used to explore this composite modification in cells31-33,36 – requiring minimal material for sensitive detection – ZUD, the first and only reagent specific for ADPrUb, remains considerably more challenging to use, requiring larger sample amounts and producing higher background33. Detection specificity was ensured using hydroxylamine, which cleaves the ester bond to release ubiquitin from mono-ADPr17,33. ZUD and mono-ADPr antibodies revealed a significant increase in DNA damage-induced ADPrUb in XRCC1 KO cells compared to WT cells (Fig. 8B), consistent with the enhanced recruitment of the ADPrUb reader RNF114 to DNA lesions (Fig. 8A). This highlights the impact of XRCC1 on ADPrUb signalling during the DNA damage response and could imply a link between XRCC1 and the various effects suggested for ADPrUb substrates including their degradation, stabilization or relocalization35,36,53.

Methods
Cell Culture and drug treatments
U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma free by ATCC cell line authentication service. Cells were cultured in GlutaMAX-DMEM supplemented with 10 % bovine serum and 100 U/ml penicillin/Streptomycin at 37 °C and 5 % CO2. hTert RPE1 cell lines and primary fibroblasts with XRCC1 mutations11 as well as control counterparts 1BR.3 (1BR) were generously provided by Dr. Keith Caldecott and confirmed mycoplasma free in in-house testing. Cells were cultured in GlutaMAX-DMEM supplemented with 10 % bovine serum and 100 U/ml penicillin/Streptomycin at 37 °C and 5 % CO2 with low-oxygen (3 %).

To induce DNA damage, the cell medium was aspirated and replaced with 37 °C complete DMEM containing 2 mM (U2OS) or 150 μM H2O2 (RPE1), 1.8 mM MMS or 15 μM CPT for indicated time points. For PARP inhibition (Olaparib, Cayman Chemical) cells were treated with 1 μM Olaparib in complete DMEM for 30 min prior to DNA damage treatment, followed by indicated H2O2 & 1 μM Olaparib treatment. For PARP1 specific inhibition cells were treated with 10 nM AZD9574 (Palacaparib, MedChemExpress) 30 min prior to DNA damage treatment, followed by indicated H2O2 & 10 nM AZD9574 treatment. For PARG inhibition cells were treated with 1 μM PARG inhibitor (PDD00017273, AdipoGen) 30 min prior to DNA damage treatment, followed by indicated H2O2 & 1 μM PARG inhibitor treatment.

For glucose starvation, cells were washed with phosphate-buffered saline (PBS) and incubated in glucose-free DMEM (L-glutamine) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin, for 3 h at 37 °C.

Plasmids
Plasmid for mEGFP-WWE (encoding the WWE domain of RNF146), was kindly provided by Dr. Sebastien Huet (University of Rennes). pmCherry-macroD2 (encoding the macrodomain of macroD2) was kindly provided by Dr. Gyula Timinszky (Biological Research Centre, Szeged, Hungary). GFP-H1.4 Plasmid was kindly provided by Dr. Ivan Ahel (Sir William Dunn School of Pathology, Oxford, England).

Antibody SpyCatcher Coupling
All Bio-Rad antibodies have been obtained unconjugated in a monovalent format and were conjugated to BiSpyCatchers in-house leading to bivalent HRP conjugated or bivalent synthetic IgG or Biotin coupled antibody formats. In brief antibody stocks were diluted to 1 mg/ml in PBS and incubated with 1/10th the volume of unconjugated antibody with BiSpyCatcher for 1 h at RT, i.e. 10 μl of BiSpyCatcher was added to 100 μl of 1 mg/ml unconjugated antibody. Afterwards they were aliquoted and stored at -20 °C.

Expression and purification of recombinant proteins
Proteins were expressed in Escherichia coli (E. coli) and purified essentially as previously described57, but with addition of a size-exclusion chromatography purification using HiLoad 16/60 Superdex 75 column. PARP1 wild-type was expressed and purified as previously reported57,58. PARG was purified as described59. HPF1 was expressed and purified as reported60.

In-vitro Modification of Proteins
Recombinant PARP1 protein (0.5 μM) was incubated with 2 mM NAD+ in the presence of HPF1 (5 μM). The reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2 and 1 ng/μL sonicated DNA. Reactions were allowed to proceed for 30 min at RT, before being stopped by the addition of 1 μM Olaparib. To create mono- and poly-ADP-ribosylated protein samples were split and one half was further incubated with 3 μM PARG for 1 h at RT and stopped by the addition of Laemmli buffer. Samples were boiled and run on an 8 % Bis-Tris gel (Sigma-Aldrich).

For Dot-blotting the same reaction as above was performed without splitting the reaction. No loading buffer was added. A dilution series of the sample was performed using reaction buffer and the same volumes (1 μl) of the probe was pipetted onto a dry nitrocellulose membrane. The membrane was left to dry and was proceeded with 5 % non-fat dried milk blocking and regular immunoblotting steps as described below.

In-vitro Modification to generate Poly-ADPr Peptides
PARP1 (PVEVVAPRGKSGAALSKKC), H3 (ARTKQTARKSTGGKAC) and H1.4 (KATGAATPKKSAKKTPKKGGK) peptide sequences were chosen based on Serine-ADPr abundances in cell-based studies. Peptide sequences were modified by adding a Cysteine for follow up Protein coupling. In separate in-vitro reactions of 250 μl, peptides (1 μg/μl) were incubated in the presence of 0.1 μM PARP1 , 5 μM HPF1 and 2 mM NAD+ for 6 h at RT, adding final 2 mM NAD+ every 2 h to the reaction. Afterwards, the reaction was split in two and half it was incubated with 3 μM PARGI for 1 h at RT. The reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl2 and 1 ng/μL sonicated DNA. Peptides were desalted and purified from recombinant proteins on in-house-manufactured SDB-XC StageTips. In brief. Stagetips were precleared using 100 % Acetone followed by isopropanol and spin until dry. Afterwards, Stagetips were activated using 100 % Methanol and spin until material still wet, followed by one wash in H2O. Peptides were loaded in H2O, followed by two washes in H2O. Peptides were eluted using 40 % ACN in 0.1 % FA and afterwards freeze-dried. Reaction without PARG was resuspended and further ADP-modified in the presence of 1.2 μM PARP1 and 2 mM NAD+ for 6 h at RT, adding final 2 mM NAD+ every 2 h to the reaction. Again, the reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl and 1 ng/μL sonicated DNA. Afterwards, peptides were desalted und purified as before and freeze-dried.

In vitro modification of mono-ADPr Peptides for Dot-Blot
PARP1 (APRGKSGAALSKKS(ph)KGQVGGK(Biotin)), H3 (1-21)(ARTKQTARKSTGGKAPRKQLAGGK(Biotin)), H3 (22-44) (ATKAARKSAPATGGVKKPHRYRPGGGK(Biotin)) and H1.4 (KATGAATPKKSAKKTPKKGKK(Biotin)) peptides, were modified for mono-ADPr as described above. In brief, peptides (1 μg/μl) were incubated in the presence of 0.1 μM PARP1, 5 μM HPF1, 3 μM PARG and 2 mM NAD+ for 6 h at RT, adding final 2 mM NAD+ every 2 h to the reaction. The reaction buffer contained 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM MgCl and 1 ng/μL sonicated DNA. Peptides were incubated with Avidin (Sigma-Aldrich, #A9275) 1:1 (w/w) for 5 min at RT. Coupled Peptides were directly pipette onto 0.2 μm Nitrocellulose membranes, left until dry and blocked with 5 % non-fat milk and incubated with antibodies as described below.

Dot-Blot with free-mono-ADPr and free-poly-ADPr
To analyze specificity of poly-ADPr specific antibodies, dot blotting was performed using Biotinylated ADPr (Jena Bioscience) and Biotin(Terminal)-Poly-ADPr (R&D Systems), which were prepared in four serial (1:2) dilutions in water. For each dot, 1 µL of prepared dilution was pipetted onto 0.2 µm Nitrocellulose membrane, left to dry around one hour RT and blocked with 5 % BSA in 1x PBS. After blocking, HRP-formats of our poly-ADPr antibodies (0.1 µg/mL) and pan-ADPr antibody (Cell Signaling Technology, 1:1000) were diluted in 1 % BSA in 1x PBS-T and the membranes were incubated for 2 h at RT and mild shaking. Membranes were then washed three times for 5 min with PBS-T. Pan-antibody was additionally incubated with secondary rabbit-HRP antibody diluted in 1 % BSA in 1x PBS-T (1:8000) for 1 h at RT and mild shaking with subsequent washing three times for 5 min with PBS-T. Membranes were developed at ChemiDoc using ECL Select Reagents.

Inverted polarity visualization technique for peptide-ADPr detection
Substrates and in-vitro ADPr modified peptides were visualized as described before54. In brief, 3 μg of peptide were mixed with TBE sample buffer and loaded on 20 % TBE gel in TBE running buffer. Samples were run with inverted polarity for 90 min at 200 V. Gels were fixed with for 30 min in 10 % Glutaraldehyde and stained with Imperial Protein Stain for 1 h after washing.

Acid–U00000000
Acid–Urea Gel Electrophoresis of ADP-ribosylated Peptides
Biotinylated poly-ADP-ribosylated histone H1 peptides (see section above) were analyzed by acid–urea (AU) gel electrophoresis based on a published protocol61, with modifications as detailed below. Where indicated, peptides were treated with recombinant PARG for 1 h at 37 °C to convert poly-ADP-ribosylation to mono-ADP-ribosylated forms. Samples were mixed with 2× Gel Loading Buffer II (Invitrogen, # AM8546G ), and up to 5 µg peptide per well was loaded.

AU gels (19:1 acrylamide:bisacrylamide) were cast at 1.5 mm thickness. Resolving gels contained 2.4 g urea, 4.5 ml 40 % acrylamide–bisacrylamide (19:1), 0.5 ml glacial acetic acid, and were filled up with water to 9.75 ml, supplemented with 200 µl 10 % APS and 40 µl TEMED. Stacking gels contained 2.4 g urea, 1.25 ml 40 % acrylamide–bisacrylamide (19:1), 0.25 ml glacial acetic acid, and were filled up with water to 4.75 ml, with 200 µl 10 % APS and 40 µl TEMED. Before adding APS and TEMED, the resolving and stacking solution were pre warmed to 30 degrees for 30 min to ensure seamless polymerization.

Gels were pre-run at 150 V with inverted polarity in 5 % acetic acid, using equilibration loading buffer (8 M urea, 10 mM Tris pH 7.5, 10 % PEG 6000) either overnight or until the current dropped below 30 mA (ca. 2 h). Samples were then run at 290 V with inverted polarity for up to 45 min in fresh 5 % acetic acid. To visualize the running front, 1 µl methyl can be added to a free pocket.

For staining, gels were fixed in 10 % glutaraldehyde for 30 min, washed 3 times with PBS, stained with Imperial Blue for 2 h, and destained overnight. For immunodetection, unfixed gels were transferred onto 0.2 µm PVDF-PSQ membranes in NuPAGE Transfer buffer with 15 % methanol at 40 V overnight at 4 °C. Membranes were crosslinked in 0.05 % glutaraldehyde for 20 min, washed in PBS three times, blocked in 5 % BSA for 1 h, and incubated with the indicated HRP-conjugated antibodies (0.0005 ug/ml) or Mono-/Poly-ADPr antibody (1 ug/ml ; #89190S, Cell Signaling Technology) for 2 h RT followed by 1 h secondary antibody incubation for uncoupled Cell Signalling Antibody. Signals were detected by chemiluminescence using Cytiva’s Amersham ECL Select Western Blotting Detection Reagent or SuperSignal West Atto Ultimate Sensitivity Substrate (Thermo Scientific).

RNA isolation and enzymatic treatment
Total RNA was isolated using the Quick-RNA Miniprep Kit (Zymo Research) according to the manufacturer's instructions and quantified by NanoDrop spectrophotometry.For digestion controls, 5 µg of RNA was incubated in 50 µL of 1× SSC buffer at 37°C for 30 min with either 1 µL RNase A/T1 mix (2 mg/mL RNase A, 5,000 U/mL RNase T1; Thermo Fisher Scientific) + 1 µL RNase I (100 U/µL; Thermo Fisher Scientific), or 1 µL Proteinase K (20 mg/mL). Digested samples and untreated controls were purified twice using the Monarch RNA Cleanup Kit (New England Biolabs) according to the manufacturer's instructions. Equal amounts of RNA from all conditions (untreated and digestion controls) were used for dot blotting. For the detection of ADP-ribosylated RNA, RNA samples (100–2,000 ng) were diluted in 200 µL of 20× SSC buffer (3M NaCl, 300mM sodium citrate, pH 7.0) and spotted onto a nitrocellulose membrane using a Bio-Dot Microfiltration Apparatus (Bio-Rad) according to the manufacturer's instructions. Membranes were air-dried for 10 min and UV-crosslinked (254 nm, 125 mJ/cm²). Membranes were washed once with PBST (PBS + 0.1% (v/v) Tween-20) for 5 min at RT with gentle shaking for the removal of the unbound RNA, then blocked in 5% (w/v) non-fat milk in PBST for 1 h at RT. Membranes were incubated overnight at 4 °C with HRP-conjugated antibodies: AbD43647 or AbD41122 (both 0.1 µg/mL) in 5% milk–PBST. After three 10-min washes with PBST, membranes were developed using enhanced chemiluminescence (ECL) reagent. Signals were acquired using a ChemiDoc MP Imaging System (Bio-Rad)

Antibody generation
Antibody generation was performed as described before21. In contrast to previously described protocol, here mono-ADPr H3S10ADPr peptides with a C-terminal cysteine (modification protocol described above) were conjugated to the carrier proteins bovine serum albumin (BSA) and human transferrin (Trf) and used for blocking during the panning process. Mono-/Poly-H3S10ADPr peptide mix with a C-terminal cysteine (modification protocol described above) were conjugated to the carrier proteins bovine serum albumin (BSA) and human transferrin (Trf) and immobilized to Maxisorp plates. After selection rounds the antibody genes were subcloned as a pool into a vector for expression of a bivalent Fab format Fab-A-F. And after expression in E. coli cultures were lysed and crude extracts were tested in ELISA, using mono-ADPr H3S10ADPr peptide, mono-ADPr PARP1S499ADPr peptide and mono-/poly-ADPr H3S10ADPr peptide mix for selection. Clones fulfilling criteria were sequenced, and the resulting unique antibodies were expressed and purified as previously described62. Purity and activity were subsequently tested by Coomassie-stained SDS-PAGE and indirect ELISA, respectively. All positive clones were validated using in-vitro modified proteins and peptides as well as with cell extracts in immunoblotting and fixed cells in immunofluorescence microscopy.

Antibody affinity maturation
Antibody affinity maturation was performed similar as described before18,63. The antibodies AbD33205 and AbD33644 were derived from the Fab phage display library HuCAL PLATINUM21,64.

For AbD33205 maturation leading to the successfully validated AbD41122 clone, we employed a selection strategy utilizing a PARP1S499-mono-ADP-ribosylated peptides (APRGKS(ADPr)GAALSKKSKGQVGGK-biotin), to preserve - and potentially enhance - the broad mono-specificity of the parental antibody.

For AbD33644 maturation leading to the successfully validated AbD55568 and AbD55558 clones, we employed a selection strategy utilizing the H3S10ADPr biotinylated peptide (ARTKQTARKS(ADPr)TGGKAPRKQLAGGK-biotin).

To identify successfully affinity-matured clones, a subset of candidates was evaluated by ELISA.

Protein Overexpression for Histone H1.4 and Live Cell Imaging
RPE1 cells were transfected with Plasmids, described above, using X-fect (Takara) according to the manufacturer's protocol. In brief, 10:3 dilutions of Plasmid:Polymer were prepared in reaction buffer and incubated for 10 min. Protein expression was performed for 48h before harvesting or imaging.

Live-Cell Imaging
For live-cell imaging experiments, cells were seeded into m-Slide 8-well polymer-bottom chambered coverslip (Ibidi), or m-Dish 35 mm polymer-bottom coverslip (Ibidi). For Hoechst presensitization, the culture medium was removed and replaced with fresh medium containing 0.3 mg/mL Hoechst 33342. Cells were incubated in this solution for 1 hour at 37 °C. Immediately before imaging, the medium was exchanged for a CO₂-independent imaging medium (Molecular Probes Live Cell Imaging Solution (Invitrogen), supplemented with 20% fetal bovine serum (FBS), 2 mM glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin).

Protein recruitment kinetics at laser-induced DNA damage sites were monitored as previously described65. Briefly, imaging was carried out using an Olympus SpinSR spinning disk confocal system equipped with a Yokogawa CSU-W1 spinning disk head (50 µm pinhole size), a UPLSAPO 100×/1.35 NA silicon-immersion objective, and a Hamamatsu ORCA-Flash4.0 sCMOS camera. Laser microirradiation was performed on Hoechst-presensitized cells along a 10 µm line across the nucleus using a continuous 405 nm laser at an intensity of 125–130 mW at the sample plane. Cells were maintained at 37 °C using a temperature-controlled chamber during imaging. Image sequences were analyzed using ImageJ/FIJI or Olympus CellSense. The irradiated region and entire nucleus were manually segmented, and the mean fluorescence intensity within the irradiated area was background-subtracted. This value was then divided by the mean nuclear intensity to correct for imaging-induced photobleaching and subsequently normalized to the pre-damage signal.

ADPrUb Immunoprecipitation
Cells were seeded in two big square dishes (500 cm2 each)/condition and treated as described before. Cells were harvested in ice cold PBS and centrifuged for 5 min, 500xg at 4 °C. Ice cold Lysis Buffer (20 mM HEPES, pH 7.0; 300 mM NaCl; 2/5 mM MgCl ; 0.5 % NP40; 20 % Glycerol, 1:1000 benzonase (750 U/ml), 20 µm Olaparib, 2 µM ADP-HPD, EDTA free Protease Inhibitor, PR619 1:1000) was added to cell pellet and incubated for 1 h at 4 °C, end-to-end rotation. Lysates were cleared by centrifugation at 16,000 xg, 5 min at 4 °C. Protein concentration was measured using Pierce Dilution-Free BCA Protein Assay Kit (#A55864, ThermoScientific). Lysate volumes corresponding to 6 mg Protein were diluted at least twice using Dilution Buffer (20 mM HEPES pH 7.0; 0.5 % NP40; 20 µm Olaparib, 2 µM ADP-HPD, EDTA free Protease Inhibitor, PR619 1:1000). 200 µg ZUD33, was coupled to Biotin BiSpyCatcher2 (BioRad, #TZC002B) according to the manufacturer’s protocol. Afterwards ZUD-Biotin was incubated with prewashed Dynabeads MyOne C1 Streptavidin beads (ThermoFisher, #65001) and incubated for 1 h at 4 °C, end-to-end rotation. Coupled beads were washed twice using NP40 containing washing buffer (20 mM HEPES pH 7.0; 300 mM NaCl, 0.05% NP40). Half of the beads were used per condition and incubated with the diluted lysates for 1 h at 4 °C, end-to-end rotation. Beads were washed 3x using NP40 containing washing buffer, followed by 3x washed NP40 free washing Buffer (20 mM HEPES pH 7.0; 300 mM NaCl). ADPrUb modifed proteins were specifically eluted using 30 µl EDTA Elution Buffer (30 mM EDTA; 20 mM HEPES pH 7.0; 300 mM NaCl). Elutions were split in two, half of the elution was treated with NH2OH (1M final concentration) for 2 h at RT. 1/10th of each sample was loaded for mono-ADPr antibodies, 9/10th of each sample was loaded for ZUD-HRP coupled detection. Samples were run on 4-12 % Bis-Tris gels (mPage, Sigma-Aldrich) and run with precooled MES running buffer (Invitrogen). Gels were transferred on PVDF membranes (Amersham) for 110 min at 110 V on ice. Membranes were blocked in 5 % non-fat dried milk for 30 min at RT, followed by overnight incubation of Antibodies/Reagents at 4 °C. Membranes were washed with PBS-T 3x 10 min before developing using ECL Select Western Blotting Detection Reagent (Amersham) or SuperSignal West Atto Ultimate Sensitivity Substrate (ThermoFisher).

ADPr Immunoprecipitation
Cells were seeded in 10 cm cell culture dishes, and treated with H2O2 as described above. Cells were harvested in ice cold PBS, and centrifuged for 5 min, 500 xg at 4 °C. Afterwards, supernatant was aspirated and cell pellet was resuspended in RIPA Buffer (50 mM HEPES, pH 7.0; 150 mM NaCl; 1 % NP40; 0.5 % Sodium deoxycholate; 0.1 % SDS). 1 µl benzonase (smDNase, 750U per sample was added and incubated for 10 min on ice. Sample were spin at max speed for 10 min and supernatant was transferred into new tube. Sample was diluted 1:3 with PBS containing ADP-HPD, Olaparib and Protease Inhibitors. 200 µg Lysate was incubated with 15 µg Antibody for 4 h at 4 °C end-to-end rotation. 15 µl prewashed Protein A (ThermoFisher) or high-capacity Streptavidin beads (ThermoFisher) were added to Lysate/Antibody mix and incubated for 1 h at RT. Beads were washed 3x with PBS. Proteins were eluted using 4x LDS Buffer supplemented with DTT at RT, second elution was performed with 4x LDS/DSTT Buffer at 95 °C.

Histone H1 Isolation
Cells were seeded on big square dishes (500 cm2), treated as described above and harvested in ice cold PBS and spun for 5 min, 500 xg at 4 °C . Cell pellet was resuspended in 500 µl 0.1 M ice cold H2SO4 and incubated for 2 h on ice. Samples were cleared by centrifugation 2,200xg for 20 min at 4 °C, resulting Pellet referred to as H2SO4 pellet. 100 µl 70 % perchloric acid was added and incubated for 1h on ice. Lysates were cleared by centrifugation 10,000 xg for 30 min at 4 °C, resulting Pellet referred to as PCA pellet. 300 µl 100 % trichloroacetic acid (TCA) was added and incubated overnight at 4 °C, samples were cleared by centrifugation at max speed for 30 min, resulting Pellet referred to as TCA pellet, this pellet contains Histone H1. All pellets were washed with 2 washes in ice cold acetone + 0.2 % HCl followed by one wash in ice cold acetone. Pellets were air dried and resuspended in H2O, Protein concentration was measured using Pierce Dilution-Free BCA Protein Assay Kit (#A55864, ThermoScientific) and samples were loaded on SDS Page gels and continued as described below.

H2SO4 Sample Preparation
RPE1 cells were treated as indicated and lysed in H2SO4, by first harvesting cells in PBS and centrifuging at 500 xg, 5 min at 4 °C. Cell pellets were resuspended in ice cold 0.1M H2SO4 and incubated for 2 h at 4 °C. Lysates were centrifuged for 20 min at 2,200 xg at 4 °C, supernatant was transferred into a fresh tube and neutralized with Tris/HCl pH 7.5. Concentrations were measured using a Pierce Dilution-Free BCA Protein Assay Kit (#A55864, ThermoScientific). Immunoblotting was performed as described below.

SDS Sample Preparation and Immunoblotting
U2OS and RPE1 cells were treated as indicated, lysed in SEDS buffer (4 % SDS; 50 mM HEPES pH 7.2, 150 mM NaCl, 5 mM MgCl2) and incubated for 5 min at RT with recombinant benzonase (smDNase, 750U per sample). Samples were centrifuged at max. speed for 10 min and supernatant was transferred into fresh tube. Sample concentration was measured using a Pierce Dilution-Free BCA Protein Assay Kit (#A55864, ThermoScientific). Samples were prepared with NuPAGE LDS sample buffer with final concentration of 5 mM DTT (Sigma) and loaded on 4-12 % Bis-Tris gel (mPage, Sigma-Aldrich). After SDS-Page the gels are transferred onto PVDF membranes (Amersham) using wet transfers at 90 mA overnight on ice. The precooled transfer buffer is 1x NuPAGE transfer buffer (Invitrogen), 20 % ethanol in water.

For immunoblotting membranes were blocked in 5 % non-fat dried milk before primary antibodies were added. Primary Antibodies (commercial antibodies and HRP-formats of Bio-Rad conjugated antibodies) were prepared in milk using concentrations found in figures. The membranes were incubated overnight at 4 °C before being washed in TBS-T (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05 % Tween-20) or PBS-T 3x10 min. Commercial antibodies were incubated with a secondary antibody (Anti-mouse IgG HRP-conjugated secondary Amersham Cat# NA931V, Anti-rabbit IgG HRP-conjugated secondary Merck Cat# GENA934-1ML) for 1 h at RT, followed by 3x10 min TBS-T or PBS-T wash. All membranes were developed using ECL Select Western Blotting Detection Reagent (Amersham). Quantification was performed in Fiji/ImageJ using rolling-ball background subtraction (radius 100 px) and fixed-size ROIs. Mean gray values were normalized as indicated, and data from three independent experiments are presented as mean ± SEM.

Immunofluorescence
U2OS and RPE1 cells were grown on sterile glass coverslips in 24-well plates. treated as indicated and fixed for 20 min with Methanol at -20 °C. The fixed cells were washed twice with PBS, and blocked for 1 h, RT with 5 % Normal-Goat-Serum in PBS supplemented with 0.3 % Triton. Primary antibodies (commercial antibodies and synthetic IgG formats of Bio-Rad conjugated antibodies) were diluted as indicated and incubated overnight at 4 °C in 1x PBS, 1 % BSA and 0.3 % Triton. Coverslips were washed in PBS 3x5 min before adding the secondary antibodies and DAPI, again in 1x PBS, 1 % BSA and 0.3 % Triton for 1 h, RT protected from light. Coverslides were washed again in PBS 3x5 min before mounted on microscopy slides using Prolong Diamond Antifade Mountant (ThermoFisher). Cells were scanned for immunofluorescence using a Leica SP8-X inverted laser-scanning confocal microscope.

For high-throughput imaging cells were grown in Ibidi 96-well glass-bottom plates, and stained as described above. After final wash cells were stored in PBS and protected from light. Cells were then imaged using a Leica Mica Microhub, confocal setting 40x objective. Data analysis was performed as described before18. In brief, raw files from microscopy were imported to Fiji. After splitting channels, the DAPI based channel was processed to generate a mask using the following filters. The mask was used to define regions of interest (ROIs) which were transferred to the ADPr fluorescence channel and mean grey values were measured. For data representation all experiments were normalized as indicated in figure legends. Means +- SEM of three biological replicates were depicted in graph. All values were imported to GraphPad Prism and plotted in a grouped data table.

ELISA
Following protocol was used to check specificity of antibody binding by ELISA: wells of the Pierce NeutrAvidin Coated Black 96-Well Plates (ThermoScientific, #15117) were first washed three times with 200 µL PBS-T (0.05 % Tween-20 in 1xPBS), then 100 µL of 61 nM peptide antigen in 1xPBS was added to each well (each antigen in duplicates per antibody tested) and incubated 1 h at RT, with mild horizontal agitation. After 1 h, peptide mix was removed and wells were blocked with 5% BSA in PBS-T for 1 h at RT, with mild horizontal agitation. Wells were washed three times with 200 µL PBS-T before primary antibodies in the monovalent format (2 µg/mL in 2.5 % BSA) were added to the wells incubated for 2 h at RT, with mild horizontal agitation. Wells were then washed five times with 200 µL PBS-T before secondary antibody (HRP-conjugated goat anti-human IgG, diluted 1:5000 in 5% BSA in PBS-T) was added to the wells and incubated 1 h at RT, with mild horizontal agitation. Wells were again washed five times with 200 µL PBS-T. To develop the signal, 100 µL of QuantaBlu mix (QuantaBlu Fluorogenic Peroxidase Substrate, # 15169, mixed according to the manufacturer instructions) was added to each well and the signal was immediately developed in microplate reader Tecan at 37 C (excitation 325 nm, emission 420 nm).

MS/MS analysis of in vitro modified peptides of H3 and H1.4
For MS/MS measurements about 20 ng of in vitro modified peptides were injected into an Oribtrap Q-Exactive HF MS (Thermo Scientific) or Orbitrap Lumos MS (Thermo Scientific) instrument connected to an EASY-nLC 1200 (Thermo Scientific) chromatographic system. 20 cm (Orbitrap Q-Exactive HF MS) or 35 cm (Orbitrap Lumos) columns were packed in CoANN 75-µm fritless emitters (MSWil, TIP36007515-20-5) with Poroshell 120-EC C18 medium (2.7 µm particle diameter). 0.1 % FA in water (Buffer A) and 0.1 % FA / 80 % ACN in water (Buffer B) were used as running buffers for chromatography. The data was collected in positive mode. The Orbitrap Lumos was equipped with a FAIMS Pro Duo interface (Thermo Scientific). The compensation voltages of the FAIMS were set to -40 V, -50 V and -60 V.

On the Orbitrap Lumos the following 60 min gradient and method was applied: 0 % to 4 % Buffer B increase in 1 min and 4 % - 31 % Buffer B in 39 min. To wash the column Buffer B was increased to 90 % over 10 min, followed by an additional 10 min wash with 90 % Buffer B. A Top20 method was used, collecting MS1 spectra from 400-1600 m/z. An AGC target of 1200000 and a maximum injection time of 50 ms was set. For MS2 scans the maximum injection time was set to 200 ms and the AGC target to 50000. The MS2 scan started at 120 m/z to include adenine diagnostic ions.

On the Q-Exactive HF MS the following 52 min gradient and method was applied for H3 peptides: 0 % to 4 % Buffer B increase in 1 min and 4 % - 31 % Buffer B in 39 min, followed by an increase of Buffer B to 50 % in 5 min. To wash the column Buffer B was increased to 90 % over 5 min, followed by an additional 2 min wash with 90 % Buffer B. A Top5 method was used, collecting MS1 spectra from 400-1600 m/z. An AGC target of 3000000 and a maximum injection time of 100 ms was set. For MS2 scans the maximum injection time was set to 200 ms and the AGC target to 1000000. The MS2 scan started at 120 m/z to include adenine diagnostic ions.

On the Q-Exactive HF MS the following 52 min gradient and method was applied for H1.4 peptides: 0 % to 20 % Buffer B increase in 45 min, followed by an increase of Buffer B to 50 % in 10 min. To wash the column Buffer B was increased to 90 % over 2 min, followed by an additional 3 min wash with 90 % Buffer B. A Top5 method was used, collecting MS1 spectra from 400-1600 m/z. An AGC target of 3000000 and a maximum injection time of 100 ms was set. For MS2 scans the maximum injection time was set to 300 ms and the AGC target to 1000000. The MS2 scan started at 120 m/z to include adenine diagnostic ions.

More details of the used methods can be accessed via the provided raw files.

MaxQuant analysis of in vitro modified peptides
Raw files of MS/MS analysis of in vitro modified H3 and H1.4 peptides were analyzed using MaxQuant66 Version 2.4.12.0.

The following settings were used for H3 peptide raw files: The maximum number of variable modifications was set to 4, with methionine oxidation, mono-ADPr on serine, Di-ADPr on serine, Tri-ADPr on serine and N-terminal acetylation allowed as variable modifications. Carbamidomethylation of cysteines was set as a fixed modification. No digest was performed, but to ensure detection of the peptide trypsin was set as a digestion enzyme and the number of missed cleavages was set to 10. A fasta containing only the H3 peptide sequence was used for the search. The minimum peptide length was set to 5 and a maximum peptide mass of 7000 was set. To also collect low quality annotated spectra the minimum score for modified peptides and the minimum delta score for modified peptides were set to 0. Second peptide search was enabled. Match between runs was also enabled with default settings. The Di-ADPr (H42 O26 P4 C30 N10) and Tri-ADPr (H63 O39 P6 C45 N15) modification was set up in MaxQuant with neutral losses corresponding to AMP, Adenosine, Adenine, ADP, ADPr, DiADPr-AMP (H28 O19 P3 C20 N5) and DiADPr-ADP (H27 O16 C20 N5 P2). The diagnostic ions described in Supplementary Figure 2 were included in the modifications. Resulting spectra were manually inspected for the presence of mono-ADPr diagnostic ions.

Three different searches with the following settings were used for H1.4 peptide raw files:
The maximum number of variable modifications was set to 4, with methionine oxidation and N-terminal acetylation always included as variable modifications. As a third modification either mono-ADPr on serine or Di-ADPr on serine or Tri-ADPr on serine were allowed as variable modifications. Carbamidomethylation of cysteines was set as a fixed modification. No digest was performed, but to ensure detection of the peptide, trypsin was set as a digestion enzyme and the number of missed cleavages was set to 10. A fasta containing only the H1.4 peptide sequence was used for the search. The minimum peptide length was set to 7 and a maximum peptide mass of 7000 was set. Identification settings were left unchanged. Second peptide search was enabled.

More details on the MaxQuant searches can be accessed via the mqpar file provided in the PXD repository.

Inspection of raw files for the presence of poly-ADPr diagnostic ions
Raw files were inspected for the presence of diagnostic ions using Freestyle. A Layout in Freestyle was created using the diagnostic ions reported in Supplementary Figure 2B. 5 ppm was set as a mass tolerance for each diagnostic ion.

MaxQuant analysis of a public data set to identify di-ADPr sites
Raw files of the proteomic data set PXD02383542 were searched for the presence of di-ADPr peptides. The raw files used for the search are labeled “20190116_LUMOS_LC4_SCL-IAH_AR_C_AF/AB1/AB2_RX_FX” in the original repository. Note that the file name either contains AF, AB1 or AB2, R values range from 1 to 4 and F values from 0 to 3, resulting in e.g. 20190116_LUMOS_LC4_SCL-IAH_AR_C_AF _R1_F0. The raw files correspond to the control group of an experiment in which ADP-ribosylated peptides were enriched using different reagents (AB1, AB2, AF) described in Figure 2A of the original publication42. MaxQuant version 2.4.12.0 was used to analyze the data. The raw files were grouped in experiments and replicates, e.g. AB11 for AB1 Replicate 1, and fractions were assigned according to the F value in the file name. 3 variable modifications were allowed per peptide. Two searches were performed, one allowed Methionine oxidation, N-terminal acetylation and mono-ADPr on S,D,E,K,C,Y,T,H,R residues and the second one allowed additional Di-ADPr on the same amino acids. Carbamidomethylation was set as a fixed modification. Trypsin was set as the digestion enzyme with 3 maximum missed cleavages. For the first search only methionine and N-terminal acetylation were allowed as variable modifications. As a fasta the human proteome (UP000005640_9606) was set. Minimum peptide length was set to 7 and maximum peptide mass to 4600. To also collect low quality annotated spectra the minimum score for modified peptides and the minimum delta score for modified peptides were set to 0. Second peptide search was enabled. Match between runs was also enabled with default settings.

Di-ADPr sites reported in Figure 2B, C and D as well as in Supplementary Figure 2 C were filtered to have a score >40, delta score >20 and a localization probability >0.8. The resulting spectra were visually inspected to check if the modification sites were covered by the sequence ions and if diagnostic ions were present. Note that MaxQuant did not annotate any diagnostic ions in EThcD spectra. Annotated Di-ADPr spectra were checked in MaxQuant and Freestyle for the presence of diagnostic ions, which are indicated in the respective figures (Figure 2, Supplementary Figure 2).

More details on the MaxQuant searches can be accessed via the mqpar file provided in the PXD repository.

Data Availability
The antibodies AbD64138 and AbD41122 generated in this study are available through Bio-Rad Laboratories. Uncoupled Fab-SpyTag (monovalent “purified” format): AbD64138ad (Bio-Rad Catalog # TZA0117): AbD41122ad (Bio-Rad Catalog # TZA0118).

Fab-SpyTag coupled to HRP-conjugated BiCatcher2 (bivalent HRP format): AbD64138pap (Bio-Rad Catalog # TZA0117P). The other antibodies can be obtained in this format via conjugation to BiCatcher2:HRP (Bio-Rad Catalog # TZC002P).

Fab-SpyTag coupled to rabbit IgG FcCatchers (IgG-like format): the antibodies can be obtained in this format via conjugation to rbIgG-FcSpyCatcher3 (Bio-Rad Catalog # TZC013). Material requests should be addressed to the corresponding author.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE67 partner repository with the dataset identifier PXD066208 [https://proteomecentral.proteomexchange.org/?pxid=PXD066208]. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Dauben et al present a large body of data that addresses a number of loosely related topics that are of current interest in the ADP-ribosylation field. These include a) the development of new and improved antibody-like reagents for the specific detection of both mono-ADPr and poly-ADPr, b) the identification of diagnostic ions that allow the discrimination between mono-ADPr and poly-ADPr modifications by mass spectrometry, c) evidence that loss of XRCC1 impacts DNA damage-induced dynamics of PARP1-dependent mono-ADP-ribosylation, poly-ADP-ribosylation and ADP-ribosyl ubiquitylation. While the overall quality of the data is excellent, and the new reagents and methodologies described here will certainly influence further studies in the field, the study is somewhat narrowly focused on a mixture of methodological advancements, so my main two main suggestions focus on expanding the biological novelty of the manuscript.\n1. Although the data in Figs 5-7 highlight the usefulness of using improved ADPr detection tools, the data on effects of XRCC1 loss on mono-ADPr, poly-ADPr and ADPr-Ub is very descriptive, without much new biological insight. While the experiments are carefully performed and demonstrate the importance of time course analyses for the study of DNA damage-induced ADPr dynamics, the findings that PARP1 activity is higher in XRCC1 KO cells and that the timing of mono vs poly-ADPr differs between different DNA damaging agents (both in WT and XRCC1 KO cells) are not particularly surprising. I strongly suggest a thorough discussion of how XRCC1 KO could be causing these differences in light of current literature, particularly ref. 8 (Demin et al Mol Cell 2021), which suggested that in MMS-treated XRCC1 KO cells, the excess PARP1 activity at early time points leads to NAD+ depletion, restricting PARP1 activity at later time points.\n2. Related to the above point, an important experiment would be to determine the impact of PARG inhibition on the formation of excess mono-ADPr in XRCC1 KO cells. This would allow a distinction between a possible role of XRCC1 in influencing direct PARP1-dependent mono-ADP-ribosylation vs XRCC1 affecting the formation of mono-ADPr as a result of a potential impact on PARG-dependent poly-ADPr hydrolysis.\nMinor comments:\n3. Sup. Fig 2 would be a little easier to interpret if the diagnostic ions in panels A and B were exactly the same and displayed in the same order. Also, it would be helpful to add an extra column in table Sup. Fig 2B indicating which ions are present for both modifications, and which are specific for poly-ADPr.\n4. The fairly frequent formation of adenosine (both from mono and poly-ADPr) indicates that cleavage at the glycosidic bond is frequent during MS fragmentation. Have the authors considered looking for other ions in which the adenosine moiety is missing?\n5. The resolution of some of the MS spectra in the files I received was insufficient to properly read the y- and b-series fragment numbering on the peptide backbones at the top of the spectra (particularly Fig. 2), or to clearly distinguish between (colored) fragment ion peak heights and the lines (are they dotted?) used to label them.\n6. Many of the western blot panels are cropped to a limited number of samples and then displayed side-by-side for comparison. This is of course necessary when different ADPr detection reagents are used (Fig. 3, 4 and Sup. Fig.4), but hampers adequate comparison between samples during a time course (Fig. 5B and Sup. Fig 5D, E).\n7. It is unclear what the “nuclear intensity” values used to plot microscopy quantifications represent. How were pixel intensities normalized to the arbitrary units in the ca. 1-100 range? Was background subtraction performed? Also, it seems that mean -+ SEM were determined as if each nucleus was an independent replicate (n is as high as 80 in some panels). This is incorrect. Instead, the values for all quantified nuclei for each biological replicate should be averaged, so that a mean intensity value for each biological replicate is obtained. These mean values per replicate are then used to derive a mean-+SEM for the experiment, with n=3.\nTypos:\n- Fig 1A, top panel – “Stragety” should be “strategy”\n- Fig. 2A, zoomed-in area of tri-ADPr peptide - ion 889.1315 label should be “AMP+ADPr”\n- Line 157 – “AMP+ADP” cannot be correct. Maybe “ADPr+ADP”?", "reviewer_2": "This work reports the development of synthetic antibodies specific for poly(ADP-ribose) using chemoenzymatically prepared peptides with varied numbers of ADP-ribose and affinity maturation of synthetic antibodies specific for mono-ADP-ribose. Then, authors used selected antibodies reagents for poly(ADP-ribose) and mono-ADP-ribose to study ADP-ribosylation derived from PARP1 in XRCC1-deficient cells. Due to technical difficulties in preparing antigenic peptides carrying poly(ADP-ribose), authors employed a different blocking strategy by utilizing peptides with mono-ADP-ribose. This way allowed identification of new antibody reagents with improved sensitivity. Through phage display-aided selection, an antibody recognizing mono-ADP-ribosylated peptides in high specificity was discovered, showing different recognition pattens from a previously published antibody for mono-ADP-ribosylation. Combining the developed antibodies for poly(ADP-ribose) and mono-ADP-ribose, including ones reported in this manuscript and one from a previous study (abd43647), authors revealed time-dependent mono- and poly-ADP-ribosylation in wild-type and XRCC1 KO cells, and an increase of ADP-ribosyl-ubiquitylation in XRCC1 KO cells upon DNA damage. The significant efforts by authors in developing and affinity maturing antibody reagents for different forms of ADP-ribosylation are commendable. But the overall technical advances in generating ADP-ribosylation-specific reagents seem incremental compared with previously published studies. While the antibody specific for poly(ADP-ribose) showed higher sensitivity, new knowledge or information revealed exclusively by this reagent is little. Likewise, a new antibody more specific for mono-ADP-ribosylation was discovered from affinity maturation, but a previously published antibody with comparable affinity and specificity could possibly address the same questions. In addition to the technical novelty and application of the reported antibodies, the findings from XRCC1 KO cells about the ADP-ribosylation dynamics and the ADP-ribosyl-ubiquitylation provide limited insights into the functions and molecular mechanisms of mono- and poly-ADP-ribosylation and composite modification related to XRCC1. Only ADP-ribosylation dynamics under different DNA damaging conditions and increased ADP-ribosyl-ubiquitylation were shown. Functional and mechanistic understandings from these observations would be critical to support the essential roles of the reported toolbox in studying XRCC1-deficiency driven ADP-ribosylation. Given the modest technical innovation, already published antibody reagents with comparable affinity and specificity, and lack of functional and mechanistic insights from the XRCC1 deficiency-related findings, this manuscript is not recommended for publication in Nature Communications.", "reviewer_3": "The new study by Dauben et al. makes an important contribution to the protein ADP-ribosylation field, particularly in terms of providing new tools for studying this modification.\nRegarding the tools, the manuscript primarily describes a strategy for selecting sensitive poly(ADP-ribose) antibodies; the development and characterisation of these antibodies for both immunoblotting and cell immunostaining applications; and the affinity-maturation of some previously developed site-specific and general mono(ADP-ribose) antibodies. The new anti-poly antibodies do appear particularly sensitive and specific.\nIn terms of biological insight, the authors apply some of these tools to show that cellular XRCC1 deficiency results in an increase in different ADP-ribosylation types including poly(ADP-ribosyl)ation, mono(ADP-ribosyl)ation, and a composite ubiquitin-mono(ADP-ribosyl) modification, the last mentioned possibly accounting for the increased accumulation of RNF114 (the reader of the composite modification in question) at the sites of laser-induced damage.\nThe study also contains further interesting observations, such as the somewhat different spatial distiribution of mono- vs poly(ADP-ribose) signals in cells (in Suppl. Fig. 3D - by the way, can the extent to which these signals are “punctate” or not be quantified?) or - this is particularly interesting - the identification of a signature di(ADP-ribose) MS fragment that is specific for poly(ADP-ribosyl)ation and seems to have confounded previous site identification efforts by mimicking a double mono(ADP-ribose).\nIt is a rigorous and interesting study and I don't have any major points of criticism. Here are some minor points that could be taken into account to further improve it:\n1) Are the new poly(ADP-ribose) antibodies specific for Ser-attached poly(ADP-ribose), or are they attachment-independent? Would they recognise Glu/Asp-linked poly(ADP-ribose)? Would they recognise free poly(ADP-ribose)? Could the authors discuss insights they might already have at their disposal that suggest answers to these questions? Would it take a lot of effort to explore this question further experimentally? I think it would be useful to at least discuss it. Similarly, do the authors think these antibodies might unequally detect chains differing in length or branching status?\n2) In terms of citations, the selection of references is generally relevant and complete, but the authors could cite the 1960s studies they mention in line 39. The statement “Dysregulation of ADPr is implicated in various diseases, including cancer and neurodegeneration, …” in lines 40-41 could also do with a citation supporting these claims. For the composite Ub-ADP-ribose modification in line 94, the Zhu et al. Sci Adv paper could be referred to alongside the cited cellular studies.\n3) The sentence in lines 55-58 (“A prime example…”) is a bit convoluted; the authors could consider simplifying it and perhaps splitting it in two.\n4) I appreciate the fact that immunoblots are generally accompanied by Ponceau S stain images serving as loading controls, but these are sometimes a bit dark and low in contrast, especially when printed out - perhaps worth checking and improving their clarity.\n5) In lines 283-285, a background signal that can be eliminated with sulphuric acid is mentioned. Could the authors comment on what this signal might be and why it gets eliminated with sulphuric acid?\n6) Could the authors explain in clearer words the term “antibody leakage” used in line 300?\n7) In line 332, could the authors clarify that they used both Olaparib and the more PARP1-specific AZD9574 to establish this conclusion?\n8) The procedure involving perchloric acid enrichment and mutation used to identify H1 as one of the bands could be briefly explained in lines 337-338. This fragment contrasts with otherwise more thorough explanations of the approaches applied.\n9) Lines 357-359: Is the more spatially heterogeneous distribution of poly(ADP-ribose) signal across cells due to the signal having already disappeared in some cells, or was it not there in some of them in the first place? Is it possible to distinguish between the two? What do the authors consider more likely?\n10) The sentence “This raises the question whether the toxic accumulation of ADP-ribosyl-linked serine ubiquitylation contributes…” sounds as if it was known that ADP-ribosyl-linked Ub was toxic, and the remaining question was whether it contributed or not to these particular symptoms. However, the accumulation of this composite modification should only be called toxic if it does cause these symptoms and this is an open question as the authors admit. In short, perhaps it’s better not to call it “toxic” until proven.\n11) Line 552: space in E. coli missing. Also, some spaces between numbers and units are missing in Methods.\n13) In Fig. 1A, it looks a bit as if the circle representing ADP-ribosyl was attached next to a serine (“S”) rather than to a serine. Maybe it is just me.\n14) Overall, the figures are high quality and clear and the legends are exhaustive, providing information on replicability, number of cells etc. The Methods description seems exhaustive.\n15) The authors could consider changing poly-ADP-ribosylation etc. to poly(ADP-ribosyl)ation - but I guess it is their conscious choice to use the hyphenated form, which I respect."}, "author_response": "Thursday, December 18, 2025\nPoint-by-point answer to Reviewers:\nNCOMMS-25-57878 (A Versatile Toolbox Reveals XRCC1 Deficiency–Driven Re-shaping of the PARP1 Mono-ADP-Ribosylation Wave)\nSummary:\nWe would like to thank the editor and the reviewers for their time in evaluating our work and for their helpful suggestions. We are encouraged by the many positive comments, and we also appreciate the specific and broader concerns that were raised. In response to these comments, we have performed substantial additional experiments and made significant revisions to the manuscript.\nBriefly, we have obtained four main sets of additional data:\n- A new main figure analyzing the impact of PARG inhibition on the formation of excess mono-ADPr in XRCC1 KO cells\n- Additional experiments demonstrating key advantages of the new tools over existing reagents: (a) Dramatic improvement in RNA ADPr detection by the new mono-ADPr antibody (data from a separate collaborative project and incorporated here); (b) detection of extremely low levels of PARP1-dependent poly-ADPr in unperturbed RPE1 cells, a signal not detectable with existing tools\n- Extensive additional characterization of the poly-ADPr antibodies using a range of poly-ADPr forms (free chains, short oligomers and Asp-/Glu-linked) to demonstrate broad substrate recognition of the new tools.\n- Strengthening of specific experiments in response to the reviewers’ concerns\nFurther, we have carefully revised the text throughout in response to the reviewers’ critique. We believe that by following reviewers’ suggestions and incorporating considerable new results and clarifications, we have significantly improved the manuscript and addressed the concerns raised. We hope that the revised version will now be suitable for publication in Nature Communications. Please find below a detailed point-by-point answer to the reviewers’ comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised version, Dauben et al addressed my concerns, including the addition of a new main figure (Fig. 7) exploring the effects of PARG inhibition on ADPr dynamics in XRCC1 KO cells, as suggested. As a result, the revised manuscript is much improved and I recommend publication. However, some minor issues still require some attention:\nSup Fig. 2A: The chemical structure for adenosine is missing on the left side of the figure, causing the chemical structures for most molecules to be misaligned with their respective profiles/labelling.\nFig. 4C: the minus (-) and plus (+) signs are presumably swapped, as glucose starvation implies lack of glucose.", "reviewer_2": "The new experiments included in the revised manuscript showed some new biological insights using the developed antibody reagents. However, a few concerns still need to be addressed to demonstrate the superiority of these antibodies over other established ones.\n1. Fig. 3B tried to evaluate affinity among various anti-poly-ADPr reagents for poly-ADPr-PARP1 at varied concentrations. The reported two synthetic antibodies were labeled with HRP molecules, but other commercial ones carrying Fc from different species would need the use of distinct secondary antibodies for detection. This experimental design cannot ensure fair comparison of their affinity and justify correlations of high signal intensities with sensitivities of primary antibody reagents.\n2. In Fig. 3A-E, 4F, and 7A, authors claimed significantly higher signals from the reported antibodies. To support these claims, quantitative analysis of the immunoblots with at least three independent replicates would be required.\n3. In Fig. 4C, the authors claimed that AbD41122 allows to detect mono-ADPr on RNA molecules. How does this affinity-matured antibody compare with other commercial ones like CST D9P7Z, MABE1016, and MABE1076 for recognition of mono-ADP ribosylated RNA?\n4. Lines 238 and 307, olaparib can inhibit PARP1 and other PARPs.\n5. The glucose label for Fig. 4C is confusing. Should be glucose starvation.", "reviewer_3": "I thank the Authors for addressing all my comments, as well as many of those raised by other reviewers.\nI particularly appreciate the new experiments exploring the specificity and sensitivity of the anti-poly antibodies, which I do think will make very useful tools. I now understand the challenges with quantifying the 'punctate' nature of the observed ADPr signals in cells, and with testing branchpoints, and I thank the Authors for explaining those. I also understand the challenge with Ponceau stain brightness, when little total protein is loaded; this is a common technical limitation.\nGiven the fact that, to me, the main value of the article lies in its methodological aspects (both in terms of antibodies and MS insights), I wonder whether the authors could still consider a different article title - one that does not highlight primarily the, somewhat secondary, biological insights, but rather both the antibodies and MS insights, presenting these as tools for monitoring PAR and MAR in different contexts. But this is just a gentle suggestion.\nOverall, I think this manuscript is ready for publication and will be useful to the community. Congratulations."}, "author_response": "Monday, February 16, 2026\nPoint-by-point answer to Reviewers:\nNCOMMS-25-57878A (Versatile and sensitive detection of mono- and poly(ADP-ribosyl)ation reveals XRCC1-dependent remodeling of PARP1 signaling)\nSummary:\nWe would like to thank the editor and the reviewers for their time and careful evaluation of our revised manuscript. We were encouraged by the indication that most points raised in the initial review had been satisfactorily addressed and the positive assessments of Reviewers #1 and #3, who now consider the manuscript suitable for publication. Below, we provide a detailed, point-by-point response to all comments on the revised manuscript. Several points relate to data already included in the original submission, while others concern a newly added experiments introduced during the revision. In all cases, we have carefully addressed the points raised and further strengthened the manuscript with substantial additional experiments.\n- We performed sets of additional experiments further comparing AbD41122 against the commercial reagents suggested by Reviewer #2 and included quantitative analysis of immunoblots\n- We further clarified the interpretation of sensitivity versus affinity in Fig. 3B and expanded the methodological explanation of the SpyTag modular system.\n- We revised the wording regarding olaparib to clearly distinguish it as a PARP inhibitor from the PARP1-specific inhibitor AZD9574.\n- Minor figure labeling issues identified by the reviewers have been corrected.\n- In response to Reviewer #3’s suggestion, we revised the title to better emphasize the methodological advances of the study.\n- The manuscript has been shortened with changes highlighted in yellow. Further reduction of the text was not feasible without compromising clarity and completeness, given the number of experiments presented and the substantial additional data and explanations requested during the two rounds of revision.\nWe believe we have significantly improved the manuscript by following reviewers’ suggestions, incorporating new results and clarifications, and addressing their concerns. We hope the revised version is now suitable for publication in Nature Communications."}]

Decision letter