In response to DNA damage, the histone PARylation factor 1 (HPF1) regulates PARP1/2 activity, facilitating serine ADP-ribosylation of chromatin-associated factors. While PARP1/2 are known for their role in DNA single-strand break repair (SSBR), the significance of HPF1 in this process remains unclear. Here, we investigated the impact of HPF1 deficiency on cellular survival and SSBR following exposure to various genotoxins. We found that HPF1 loss did not generally increase cellular sensitivity to agents that typically induce DNA single-strand breaks (SSBs) repaired by PARP1. SSBR kinetics in HPF1-deficient cells were largely unaffected, though its absence partially influenced the accumulation of SSB intermediates after exposure to specific genotoxins in certain cell lines, likely due to altered ADP-ribosylation of chromatin. Despite reduced serine mono-ADP-ribosylation, HPF1-deficient cells maintained robust poly-ADP-ribosylation at SSB sites, possibly reflecting PARP1 auto-poly-ADP-ribosylation at non-serine residues. Notably, poly-ADP-ribose chains were sufficient to recruit the DNA repair factor XRCC1, which may explain the relatively normal SSBR capacity in HPF1-deficient cells. These findings suggest that HPF1 and histone serine ADP-ribosylation are largely dispensable for PARP1-dependent SSBR in response to genotoxic stress, highlighting the complexity of mechanisms that maintain genomic stability and chromatin remodeling.

• MeSH
• Cell Line MeSH
• Chromatin metabolism   MeSH
• DNA-Binding Proteins metabolism   genetics   MeSH
• Histones metabolism   MeSH
• Nuclear Proteins metabolism   genetics   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• DNA Repair * MeSH
• Poly ADP Ribosylation MeSH
• Poly (ADP-Ribose) Polymerase-1 * metabolism   genetics   MeSH
• Poly(ADP-ribose) Polymerases metabolism   genetics   MeSH
• X-ray Repair Cross Complementing Protein 1 metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH
• DNA-Binding Proteins MeSH
• Histones MeSH
• HPF1 protein, human MeSH Browser
• Nuclear Proteins MeSH
• PARP1 protein, human MeSH Browser
• Poly (ADP-Ribose) Polymerase-1 * MeSH
• Poly(ADP-ribose) Polymerases MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

• Keywords
• BRCA2, DNA Repair, EXO1, FEN1, PARG,
• MeSH
• Flap Endonucleases genetics   metabolism   therapeutic use   MeSH
• DNA Repair Enzymes genetics   MeSH
• Exodeoxyribonucleases genetics   MeSH
• Glycoside Hydrolases genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Neoplasms * drug therapy   genetics   MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors pharmacology   MeSH
• DNA Damage MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flap Endonucleases MeSH
• DNA Repair Enzymes MeSH
• EXO1 protein, human MeSH Browser
• Exodeoxyribonucleases MeSH
• FEN1 protein, human MeSH Browser
• Glycoside Hydrolases MeSH
• Tumor Suppressor Protein p53 * MeSH
• Poly(ADP-ribose) Polymerase Inhibitors MeSH

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1-/- mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1-/- cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

• MeSH
• DNA genetics   MeSH
• Transcription, Genetic genetics   MeSH
• Histones metabolism   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• DNA Repair genetics   MeSH
• Oxidative Stress genetics   MeSH
• Hydrogen Peroxide toxicity   MeSH
• Poly (ADP-Ribose) Polymerase-1 genetics   metabolism   MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   metabolism   MeSH
• Ubiquitin-Specific Proteases metabolism   MeSH
• Ubiquitination physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Histones MeSH
• PARP1 protein, human MeSH Browser
• Hydrogen Peroxide MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• Ubiquitin-Specific Proteases MeSH
• USP3 protein, human MeSH Browser
• XRCC1 protein, human MeSH Browser