RESUMEN
MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi.
RESUMEN
DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and differentiated cells. Using single-cell microscopic approaches, we map the spatio-temporal genome replication as a function of chromatin marks/compaction level. Furthermore, we mapped the replication timing of subchromosomal tandem repeat regions and interspersed repeat sequence elements. Albeit the majority of these genomic repeats did not change their replication timing from pluripotent to differentiated cells, we found developmental changes in the replication timing of rDNA repeats. Comparing single-cell super-resolution microscopic data with data from genome-wide sequencing approaches showed comparable numbers of replicons and large overlap in origins numbers and genomic location among developmental states with a generally higher origin variability in pluripotent cells. Using ratiometric analysis of incorporated nucleotides normalized per replisome in single cells, we uncovered differences in fork speed throughout the S phase in pluripotent cells but not in somatic cells. Altogether, our data define similarities and differences on the replication program and characteristics in human cells at different developmental states.
Asunto(s)
Cromatina , Genoma , Humanos , Cromatina/genética , Momento de Replicación del ADN , Fase S , Replicación ViralRESUMEN
The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless-Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless-Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.
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Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the distribution of origin of recognition complex (ORC) binding sites by ChIP-seq of two PfORC subunits and mapped active ORIs using NFS and SNS-seq. We show that ORIs lack sequence specificity but are not randomly distributed, and group in clusters. Licensing is biased towards regions of higher GC content and associated with G-quadruplex forming sequences (G4FS). While strong transcription likely enhances firing, active origins are depleted from transcription start sites. Instead, most accumulate in transcriptionally active gene bodies. Single molecule analysis of nanopore reads containing multiple initiation events, which could have only come from individual nuclei, showed a relationship between the replication fork pace and the distance to the nearest origin. While some similarities were drawn with the canonic eukaryote model, the distribution of ORIs in P. falciparum is likely shaped by unique genomic features such as extreme AT-richness-a product of evolutionary pressure imposed by the parasitic lifestyle.
Asunto(s)
Plasmodium falciparum , Origen de Réplica , Humanos , Sitios de Unión , Mapeo Cromosómico , Replicación del ADN , Genómica , Plasmodium falciparum/genética , Origen de Réplica/genética , Transcripción GenéticaRESUMEN
Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.
Asunto(s)
Cromatina , Replicación del ADN , Humanos , Fase S , Ciclo Celular , ADN HelicasasRESUMEN
In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.
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Proteína Potenciadora del Homólogo Zeste 2 , Histonas , Animales , Histonas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Cromatina , Replicación del ADN/genética , ADNRESUMEN
BACKGROUND: Urinary tract infestation by Capillaria spp. in domestic cats is rather rare, but can cause clinical symptoms and affect behaviour. To our knowledge, this report is the first to describe a case of urinary capillariosis in a cat in Poland. CASE PRESENTATION: A female formerly stray cat aged about 1.5 years showing dysuria, stranguria, periuria and lethargy was presented at the veterinary clinic. Urinalysis revealed the presence of Capillaria plica eggs in the sediment. The cat was treated successfully with three topical doses of Broadline (Merial, Toulouse, France). CONCLUSIONS: C. plica is a nematode whose definitive hosts are carnivores, which are infected by eating earthworms (the intermediate hosts). Thus, C. plica infestation is more frequent in wild carnivores and dogs, and rare in cats. Symptomatic bladder capillariosis in cats is very rarely diagnosed and described.
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Enfermedades de los Gatos , Infecciones por Enoplida , Animales , Gatos , Femenino , Capillaria , Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/tratamiento farmacológico , Disuria/veterinaria , Infecciones por Enoplida/diagnóstico , Infecciones por Enoplida/tratamiento farmacológico , Infecciones por Enoplida/veterinaria , Óvulo , PoloniaRESUMEN
It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA's genome integrity. Cosmic radiation due to Earth's weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil-DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth's atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.
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ADN Glicosilasas/metabolismo , Reparación del ADN , Evolución Molecular , Oxígeno/metabolismo , Alquilación , Animales , Daño del ADN , HumanosRESUMEN
To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.
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Replicación del ADN/genética , Heterocromatina/genética , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Animales , Diferenciación Celular/genética , Centrómero/genética , Duplicación Cromosómica/genética , Cromosomas Humanos Y/genética , Genoma/genética , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Fase S/genética , Imagen Individual de MoléculaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Biocatálisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Humanos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.
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Replicación del ADN/genética , G-Cuádruplex , Mamíferos/genética , Origen de Réplica/genética , Animales , Células Cultivadas , Vectores Genéticos/genética , Humanos , Ratones , Mutación , Células 3T3 NIH , Oocitos/metabolismo , Plásmidos/genética , Xenopus laevisRESUMEN
DNA replication starts with the opening of DNA at sites called DNA replication origins. From the single sequence-specific DNA replication origin of the small Escherichia coli genome, up to thousands of origins that are necessary to replicate the large human genome, strict sequence specificity has been lost. Nevertheless, genome-wide analyses performed in the recent years, using different mapping methods, demonstrated that there are precise locations along the metazoan genome from which replication initiates. These sites contain relaxed sequence consensus and epigenetic features. There is flexibility in the choice of origins to be used during a given cell cycle, probably imposed by evolution and developmental constraints. Here, we will briefly describe their main features.
Asunto(s)
Replicación del ADN , Origen de Réplica , Animales , Ciclo Celular , Epigénesis Genética , Estudio de Asociación del Genoma Completo , Humanos , Motivos de Nucleótidos , Levaduras/química , Levaduras/genéticaRESUMEN
Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.
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Replicación del ADN , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Humanos , MetilaciónRESUMEN
AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(É)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of É-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three É-adducts, 1,N(6)-ethenoadenine (ÉA), 3,N(4)-ethenocytosine (ÉC) and 1,N(2)-ethenoguanine (1,N(2)-ÉG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for É-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ÉA and ÉC from ds and ssDNA but were inactive toward 1,N(2)-ÉG. SC-1A repaired only ÉA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three É-adducts in dsDNA, while only ÉA and ÉC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ÉC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ÉG and that ALKBH3 removes only ÉC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.
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Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Aductos de ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Dioxigenasas/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Bacterias/genética , Citosina/análogos & derivados , Citosina/metabolismo , ADN/metabolismo , ADN Glicosilasas/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Oxigenasas de Función Mixta/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Rhizobium etli/enzimología , Rhizobium etli/genética , Streptomyces/enzimología , Streptomyces/genética , Especificidad por Sustrato , Xanthomonas campestris/enzimología , Xanthomonas campestris/genéticaRESUMEN
BACKGROUND: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized. METHODOLOGY/PRINCIPAL FINDINGS: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' â 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 µM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. CONCLUSIONS/SIGNIFICANCE: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Homología de Secuencia de Ácido Nucleico , Triticum/enzimología , Triticum/genética , Secuencia de Aminoácidos , Biocatálisis , Clonación Molecular , ADN/química , ADN/genética , ADN/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
Spontaneous hydrolytic deamination of cytosine to uracil (U) in DNA is a constant source of genome instability in cells. This mutagenic process is greatly enhanced at high temperatures and in single-stranded DNA. If not repaired, these uracil residues give rise to C â T transitions, which are the most common spontaneous mutations occurring in living organisms and are frequently found in human tumors. In the majority of species, uracil residues are removed from DNA by specific uracil-DNA glycosylases in the base excision repair pathway. Alternatively, in certain archaeal organisms, uracil residues are eliminated by apurinic/apyrimidinic (AP) endonucleases in the nucleotide incision repair pathway. Here, we characterized the substrate specificity of the major human AP endonuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligonucleotide duplexes containing a single Uâ G base pair; this activity depends strongly on the sequence context and the base opposite to U. The apparent kinetic parameters of the reactions show that APE1 has high affinity for DNA containing U but cleaves the DNA duplex at an extremely low rate. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of a Uâ G duplex generates the expected DNA fragments containing a 5'-terminal deoxyuridine monophosphate. The fact that U in duplex DNA is recognized and cleaved by APE1 in vitro suggests that this property of the exonuclease III family of AP endonucleases is remarkably conserved from Archaea to humans. We propose that nucleotide incision repair may act as a backup pathway to base excision repair to remove uracils arising from cytosine deamination.
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Reparación del ADN , ADN/metabolismo , Nucleótidos/metabolismo , Transducción de Señal , Uracilo/metabolismo , Secuencia de Bases , Biocatálisis , Línea Celular , Citosina/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Desaminación , Humanos , Cinética , Methanosarcina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Sulfitos , Timina ADN Glicosilasa/metabolismoRESUMEN
BACKGROUND: Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5' to a damaged base in a DNA glycosylase-independent manner. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC(50) values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εAâ¢T and εCâ¢G duplexes generates, as expected, DNA fragments containing 5'-terminal ε-base residue. CONCLUSIONS/SIGNIFICANCE: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.
Asunto(s)
Aductos de ADN/metabolismo , Reparación del ADN , Mutágenos/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Biocatálisis , Extractos Celulares , Sistema Libre de Células , Citosina/análogos & derivados , Citosina/metabolismo , Aductos de ADN/química , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Células HeLa , Humanos , Cinética , Oligonucleótidos/metabolismo , Oxidación-Reducción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Timina/análogos & derivados , Timina/metabolismo , Factores de TiempoRESUMEN
One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>Câ«A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA(-)Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II. Mutation spectrum established for strains expressing only Pol V, showed that in uvrA(-) bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:TâC:G, A:TâG:C, G:CâA:T and G:CâT:A prevailed. The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.
Asunto(s)
Aldehídos/toxicidad , Bacteriófago M13/genética , Daño del ADN/efectos de los fármacos , ADN Polimerasa II/genética , ADN Polimerasa beta/genética , Peroxidación de Lípido , Bacteriófago M13/metabolismo , Secuencia de Bases , Aductos de ADN/genética , Aductos de ADN/metabolismo , ADN Polimerasa II/metabolismo , ADN Polimerasa beta/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Operón Lac/genética , Datos de Secuencia Molecular , Mutagénesis/efectos de los fármacos , Tasa de Mutación , Mutación Puntual , Respuesta SOS en GenéticaRESUMEN
One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48% of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA(-) strain were G:C --> T:A transversions, occurring within the sequence which in recA(+) strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C --> A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.