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
• buněčné linie MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny metabolismus   genetika   MeSH
• histony metabolismus   MeSH
• jaderné proteiny metabolismus   genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• oprava DNA * MeSH
• poly-ADP-ribosylace MeSH
• poly(ADP-ribosa)polymerasa 1 * metabolismus   genetika   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   genetika   MeSH
• protein XRCC1 metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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.

• MeSH
• DNA MeSH
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• oprava DNA MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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 genetika   MeSH
• genetická transkripce genetika   MeSH
• histony metabolismus   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oprava DNA genetika   MeSH
• oxidační stres genetika   MeSH
• peroxid vodíku toxicita   MeSH
• poly(ADP-ribosa)polymerasa 1 genetika   metabolismus   MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• specifické proteázy ubikvitinu metabolismus   MeSH
• ubikvitinace fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasa ATP metabolismus   MeSH
• DNA-polymerasa beta metabolismus   MeSH
• DNA genetika   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• jednořetězcové zlomy DNA MeSH
• lidé MeSH
• oprava DNA účinky léků   genetika   MeSH
• PARP inhibitory farmakologie   MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• poškození DNA účinky léků   genetika   MeSH
• protein XRCC1 metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural 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
• apraxie genetika   patologie   MeSH
• Charcotova-Marieova-Toothova nemoc genetika   patologie   MeSH
• dvouřetězcové zlomy DNA * MeSH
• enzymy opravy DNA genetika   MeSH
• fibroblasty metabolismus   patologie   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• mikrocefalie genetika   patologie   MeSH
• mutace genetika   MeSH
• oprava DNA genetika   MeSH
• protein XRCC1 genetika   MeSH
• záchvaty genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.

• MeSH
• adenosindifosfát ribosa chemie   MeSH
• chromatin chemie   MeSH
• fibroblasty MeSH
• genový knockout MeSH
• glykosidhydrolasy genetika   MeSH
• HEK293 buňky MeSH
• histony chemie   MeSH
• jednořetězcové zlomy DNA * MeSH
• lidé MeSH
• mutace * MeSH
• nádorové buněčné linie MeSH
• neurodegenerativní nemoci genetika   MeSH
• oprava DNA * MeSH
• protein XRCC1 genetika   MeSH
• regulace genové exprese MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human gene that encodes XRCC1 was cloned nearly thirty years ago but experimental analysis of this fascinating protein is still unveiling new insights into the DNA damage response. XRCC1 is a molecular scaffold protein that interacts with multiple enzymatic components of DNA single-strand break repair (SSBR) including DNA kinase, DNA phosphatase, DNA polymerase, DNA deadenylase, and DNA ligase activities that collectively are capable of accelerating the repair of a broad range of DNA single-strand breaks (SSBs). Arguably the most exciting aspect of XRCC1 function that has emerged in the last few years is its intimate relationship with PARP1 activity and critical role in preventing hereditary neurodegenerative disease. Here, I provide an update on our current understanding of XRCC1, and on the impact of hereditary mutations in this protein and its protein partners on human disease.

• MeSH
• DNA metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• jednořetězcové zlomy DNA MeSH
• lidé MeSH
• oprava DNA * MeSH
• protein XRCC1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: We aimed to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with myelodysplastic syndrome (MDS). METHODS: Targeted enrichment resequencing of 84 DNA repair genes was initially performed on a screening cohort of MDS patients. Real-time polymerase chain reaction was used for genotyping selected SNPs in the validation cohort of patients. RESULTS: A heterozygous frameshift mutation in the XRCC2 gene was identified. It leads to the formation of a truncated non-functional protein and decreased XRCC2 expression level. Decreased expression levels of all DNA repair genes functionally connected with mutated XRCC2 were also present. Moreover, a synonymous substitution in the PRKDC gene and 2 missense mutations in the SMUG1 and XRCC1 genes were also found. In the screening cohort, 6 candidate SNPs were associated with the tendency to develop MDS: rs4135113 (TDG, p = 0.03), rs12917 (MGMT, p = 0.003), rs2230641 (CCNH, p = 0.01), rs2228529 and rs2228526 (ERCC6, p = 0.04 and p = 0.03), and rs1799977 (MLH1, p = 0.04). In the validation cohort, only a polymorphism in MLH1 was significantly associated with development of MDS in patients with poor cytogenetics (p = 0.0004). CONCLUSION: Our study demonstrates that genetic variants are present in DNA repair genes of MDS patients and may be associated with susceptibility to MDS.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• genetická predispozice k nemoci MeSH
• jaderné proteiny genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• MutL homolog 1 genetika   MeSH
• myelodysplastické syndromy enzymologie   genetika   metabolismus   MeSH
• oprava DNA * MeSH
• protein XRCC1 genetika   MeSH
• proteinkinasa aktivovaná DNA genetika   MeSH
• uracil-DNA-glykosidasa genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.

• MeSH
• "flap" endonukleasy metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasa ATP metabolismus   MeSH
• DNA genetika   metabolismus   MeSH
• lidé MeSH
• oprava DNA MeSH
• poly(ADP-ribosa)polymerasa 1 metabolismus   MeSH
• poly(ADP-ribosa)polymerasy genetika   metabolismus   MeSH
• polyadenosindifosfátribosa metabolismus   MeSH
• poškození DNA MeSH
• protein XRCC1 metabolismus   MeSH
• replikace DNA fyziologie   MeSH
• S fáze fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH