Flap endonuclease 1 (FEN1)-dependent long-patch repair has been considered a minor sub-pathway of DNA single-strand break repair (SSBR), activated only when short-patch repair is not feasible. However, the significance of long-patch repair in living cells remains unclear. Here, we employed human RPE-1 cells with FEN1 deletion to compare the requirements for short- and long-patch pathways for the rapid repair of various types of DNA single-strand breaks (SSBs). We found that SSBs arising from abortive topoisomerase 1 activity are repaired efficiently without FEN1. In contrast, the rapid repair of SSBs arising during base excision repair following treatment with methyl methanesulphonate (MMS) or following treatment with hydrogen peroxide (H2O2) exhibits an unexpectedly high dependence on FEN1. Indeed, in G1 phase, FEN1 deletion slows the rate of SSBR to a similar or even greater extent than deletion of the short-patch repair proteins XRCC1 or POLβ. As expected, the combined deletion of FEN1 with XRCC1 or POLβ has an additive or synergistic effect, severely attenuating SSBR rates after MMS or H2O2 exposure. These data highlight an unanticipated requirement for FEN1 in the rapid repair of SSBs in human cells, challenging the prevailing view that long-patch repair is a minor sub-pathway of SSBR.

• MeSH
• Flap Endonucleases * genetics   physiology   metabolism   MeSH
• Cell Line MeSH
• DNA-Binding Proteins genetics   MeSH
• DNA Topoisomerases, Type I metabolism   MeSH
• G1 Phase * genetics   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• Methyl Methanesulfonate toxicity   MeSH
• DNA Repair * MeSH
• Hydrogen Peroxide pharmacology   toxicity   MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flap Endonucleases * MeSH
• DNA-Binding Proteins MeSH
• DNA Topoisomerases, Type I MeSH
• FEN1 protein, human MeSH Browser
• Methyl Methanesulfonate MeSH
• Hydrogen Peroxide MeSH
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

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

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

• Keywords
• BRCA1, BRCA2, CP: Molecular biology, double-strand break, pluripotency, replication gap suppression, replication stress, somatic cell reprogramming, stalled replication fork,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 * metabolism   genetics   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Humans MeSH
• Mice MeSH
• DNA Repair * MeSH
• Cellular Reprogramming * MeSH
• BRCA1 Protein * metabolism   genetics   MeSH
• Recombinational DNA Repair MeSH
• DNA Replication MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 * MeSH
• BRCA1 protein, human MeSH Browser
• Brca1 protein, mouse MeSH Browser
• BRCA1 Protein * MeSH
• TP53BP1 protein, human MeSH Browser
• Trp53bp1 protein, mouse 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

DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.

• MeSH
• Chromatin MeSH
• DNA genetics   MeSH
• Emetine * pharmacology   MeSH
• DNA, Single-Stranded * MeSH
• DNA Replication MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Emetine * MeSH
• DNA, Single-Stranded * MeSH
• Okazaki fragments MeSH Browser

Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.

• MeSH
• DNA genetics   MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• Poly (ADP-Ribose) Polymerase-1 genetics   MeSH
• DNA Damage MeSH
• DNA Replication * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

• Keywords
• DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
• MeSH
• DNA MeSH
• Endothelial Cells metabolism   MeSH
• Histones * metabolism   MeSH
• Induced Pluripotent Stem Cells * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• DNA Repair MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Histones * MeSH
• MACROH2A1 protein, human MeSH Browser
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1-/- cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.

• Keywords
• PARP inhibitors, PARP trapping, PARP1, XRCC1 protein complexes, base excision repair, single-strand breaks,
• MeSH
• Cell Line MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA Ligase ATP metabolism   MeSH
• DNA Polymerase beta metabolism   MeSH
• DNA genetics   MeSH
• Fibroblasts drug effects   metabolism   MeSH
• DNA Breaks, Single-Stranded MeSH
• Humans MeSH
• DNA Repair drug effects   genetics   MeSH
• Poly(ADP-ribose) Polymerase Inhibitors pharmacology   MeSH
• Poly (ADP-Ribose) Polymerase-1 metabolism   MeSH
• Poly(ADP-ribose) Polymerases metabolism   MeSH
• DNA Damage drug effects   genetics   MeSH
• X-ray Repair Cross Complementing Protein 1 metabolism   MeSH
• Protein Binding drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA Ligase ATP MeSH
• DNA Polymerase beta MeSH
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors 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

Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1Nes-Cre ) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease.

• Keywords
• DNA strand break, XRCC1, neurodegeneration, poly(ADP-ribose) polymerase, seizures,
• MeSH
• DNA-Binding Proteins * genetics   metabolism   MeSH
• DNA MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• DNA Repair genetics   MeSH
• Poly (ADP-Ribose) Polymerase-1 genetics   metabolism   MeSH
• Calcium * MeSH
• Seizures genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• DNA MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH
• Calcium * MeSH

Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.

• MeSH
• Apraxias genetics   pathology   MeSH
• Charcot-Marie-Tooth Disease genetics   pathology   MeSH
• DNA Breaks, Double-Stranded * MeSH
• DNA Repair Enzymes genetics   MeSH
• Fibroblasts metabolism   pathology   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   MeSH
• DNA Breaks, Single-Stranded * MeSH
• Humans MeSH
• Microcephaly genetics   pathology   MeSH
• Mutation genetics   MeSH
• DNA Repair genetics   MeSH
• X-ray Repair Cross Complementing Protein 1 genetics   MeSH
• Seizures genetics   pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Repair Enzymes MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• PNKP protein, human MeSH Browser
• X-ray Repair Cross Complementing Protein 1 MeSH
• XRCC1 protein, human MeSH Browser