single-strand breaks
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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
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
- MeSH
- 5-methylcytosin metabolismus MeSH
- buněčné linie MeSH
- DNA biosyntéza MeSH
- jednořetězcové zlomy DNA * MeSH
- lidé MeSH
- metylace MeSH
- neurony metabolismus MeSH
- oprava DNA * MeSH
- poly(ADP-ribosa)polymerasy metabolismus MeSH
- replikace DNA MeSH
- sekvenční analýza DNA MeSH
- zesilovače transkripce genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural 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
DNA strand breaks arise continuously as the result of intracellular metabolism and in response to a multitude of genotoxic agents. To overcome such challenges to genomic stability, cells have evolved genome surveillance pathways that detect and repair damaged DNA in a coordinated fashion. Here we identify the previously uncharacterized human protein Xip1 (C2orf13) as a novel component of the checkpoint response to DNA strand breaks. Green fluorescent protein-tagged Xip1 was rapidly recruited to sites of DNA breaks, and this accumulation was dependent on a novel type of zinc finger motif located in the C terminus of Xip1. The initial recruitment kinetics of Xip1 closely paralleled that of XRCC1, a central organizer of single strand break (SSB) repair, and its accumulation was both delayed and sustained when the detection of SSBs was abrogated by inhibition of PARP-1. Xip1 and XRCC1 stably interacted through recognition of CK2 phosphorylation sites in XRCC1 by the Forkhead-associated (FHA) domain of Xip1, and XRCC1 was required to maintain steady-state levels of Xip1. Moreover, Xip1 was phosphorylated on Ser-116 by ataxia telangiectasia-mutated in response to ionizing radiation, further underscoring the potential importance of Xip1 in the DNA damage response. Finally, depletion of Xip1 significantly decreased the clonogenic survival of cells exposed to DNA SSB- or double strand break-inducing agents. Collectively, these findings implicate Xip1 as a new regulator of genome maintenance pathways, which may function to organize DNA strand break repair complexes at sites of DNA damage.
- MeSH
- ATM protein MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA-lyasa (apurinová nebo apyrimidinová) MeSH
- dvouřetězcové zlomy DNA * MeSH
- fosfoproteiny genetika metabolismus MeSH
- fosforylace MeSH
- jednořetězcové zlomy DNA * MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nestabilita genomu * fyziologie MeSH
- oprava DNA * fyziologie MeSH
- posttranslační úpravy proteinů fyziologie MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- terciární struktura proteinů genetika MeSH
- zinkové prsty genetika MeSH
- Check Tag
- lidé MeSH
We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m3) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 1.5 to 4.2 +/- 0.8).
- MeSH
- aplikace inhalační MeSH
- buňky kostní dřeně cytologie MeSH
- butadieny aplikace a dávkování krev toxicita MeSH
- deoxyribonukleasa (pyrimidinový dimer) metabolismus MeSH
- erytrocyty cytologie MeSH
- financování organizované MeSH
- hepatocyty metabolismus MeSH
- kometový test MeSH
- mikrojádra chromozomálně defektní MeSH
- myši MeSH
- oprava DNA MeSH
- poškození DNA MeSH
- proteiny z Escherichia coli metabolismus MeSH
- regresní analýza MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
