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1.
Nucleic Acids Res ; 51(12): 6321-6336, 2023 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-37216593

RESUMEN

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.


Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Animales , Humanos , Virus de la Fiebre Porcina Africana/metabolismo , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Péptidos , Saccharomyces cerevisiae/metabolismo , Porcinos , ADN Polimerasa beta/metabolismo
2.
Chem Rec ; 24(2): e202300262, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37850545

RESUMEN

Merocyanines, thanks to their easily adjustable electronic structure, appear to be the most versatile and promising functional dyes. Their D-π-A framework offers ample opportunities for custom design through variations in both donor/acceptor end-groups and the π-conjugated polymethine chain, and leads to a broad range of practical properties, including noticeable solvatochromism, high polarizability/hyperpolarizabilities, and the ability to sensitize various physicochemical processes. Accordingly, merocyanines are applied and extensively studied in various fields, such as light-converting materials for optoelectronics, nonlinear optics, optical storage, solar cells, fluorescent probes, and antitumor agents in photodynamic therapy. This review encompasses both classical and novel more important publications on the structure-property relationships in merocyanines, with particular emphasis on the results by A.  I. Kiprianov and his followers in Institute of Organic Chemistry in Kyiv, Ukraine.

3.
Int J Mol Sci ; 25(16)2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39201583

RESUMEN

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.


Asunto(s)
División del ADN , Pyrococcus furiosus , Pyrococcus furiosus/enzimología , Cinética , Proteínas Arqueales/metabolismo , Proteínas Arqueales/química , Especificidad por Sustrato , Conformación de Ácido Nucleico , ADN/metabolismo
4.
Phys Chem Chem Phys ; 25(34): 22851-22861, 2023 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-37584652

RESUMEN

The effect of localized surface plasmon resonance (LSPR) of a system consisting of a highly dipolar merocyanine dye and a silver nanoparticle (NP) was studied experimentally and theoretically. A theoretical model for estimating the fluorescence quantum yield (φfl) using quantum chemical calculations of intramolecular and intermolecular electronic transition rate constants was developed. Calculations show that the main deactivation channels of the lowest excited singlet state of the studied merocyanines are internal conversion (kIC(S1 → S0)) and fluorescence (kr(S1 → S0)). The intersystem-crossing transition has a low probability due to the large energy difference between the singlet and triplet levels. In the presence of plasmonic NPs, the fluorescence quantum yield is increased by a factor of two according to both experiment and computations. The calculated values of φfl, when considering changes in kr(S1 → S0) and the energy-transfer rate constant (ktransfer) from the dye to the NP was also twice as large at distances of 6-8 nm between the NP and the dye molecule. We also found that the LSPR effect can be increased or decreased depending on the value of the dielectric constant (εm) of the environment.

5.
Int J Mol Sci ; 24(11)2023 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-37298543

RESUMEN

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polß's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polß efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polß's activity toward the model DNA intermediates.


Asunto(s)
ADN Glicosilasas , ADN Polimerasa beta , Humanos , ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN Glicosilasas/metabolismo , Replicación del ADN , ADN , Daño del ADN
6.
Int J Mol Sci ; 23(22)2022 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-36430884

RESUMEN

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5' side of an AP site, thereby forming a single-strand break containing 3'-OH and 5'-dRP ends. In addition, Apn2 has 3'-phosphodiesterase activity (removing 3'-blocking groups) and 3' → 5' exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3'-5'-exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single-strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped-flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5' overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate-limiting step in the enzymatic reaction. We determined an influence of the nature of the 3'-terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context-specific.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Saccharomyces cerevisiae , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Cinética , Endonucleasas , Exonucleasas
7.
Int J Mol Sci ; 23(5)2022 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-35270011

RESUMEN

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5' to an AP-site; can recognize and process some damaged nucleosides; and possess 3'-phosphodiesterase, 3'-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions' concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3'-5' exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.


Asunto(s)
Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Endorribonucleasas/metabolismo , Modelos Moleculares , Especificidad por Sustrato
8.
Molecules ; 27(15)2022 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-35956910

RESUMEN

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.


Asunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Reparación del ADN , Proteínas de Escherichia coli , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , ADN/química , Reparación del ADN/fisiología , Dioxigenasas/genética , Dioxigenasas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Conformación Proteica , Espectrometría de Fluorescencia
9.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-34445579

RESUMEN

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site-containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes' intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.


Asunto(s)
División del ADN , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Nucleótidos/metabolismo , Sitios de Unión , Dominio Catalítico , Reparación del ADN , Escherichia coli , Humanos , Cinética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Nucleótidos/química , Conformación Proteica , Especificidad por Sustrato
10.
Phys Chem Chem Phys ; 22(5): 2748-2762, 2020 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-31950958

RESUMEN

Three vinylogous series of merocyanines with the tricyanofuran (TCF) acceptor and different electron-donor groups have been synthesized, and their absorption and fluorescence spectra have been studied in different polarity solvents to reveal the polyene-polymethine transitions of their electronic structures. The TCF dyes with the indole or benzimidazole donor groups have relatively bright fluorescence in the red and near-IR spectral ranges. From the obtained data, the electron-acceptor ability of the TCF group has been estimated to be close to that of the thiobarbituric residue, while the TCF group is less prone to formation of solute-solvent H-bonds in protic media. From the DFT and TD-DFT calculations of the TCF-based dyes, performed using B3LYP, CAM-B3LYP, and M06-2X hybrid functionals, it has been revealed that their two major conformers have very close spectral-fluorescence properties, which explains why they are undistinguishable in the steady-state electronic spectra. However, the calculated polarizabilities and the first hyperpolarizabilities are more dependent on the molecular geometry, with both parameters being greater for the transoid conformer.

11.
Nucleic Acids Res ; 46(5): 2417-2431, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29361132

RESUMEN

Poly(ADP-ribose) polymerases (PARPs) act as DNA break sensors and catalyze the synthesis of polymers of ADP-ribose (PAR) covalently attached to acceptor proteins at DNA damage sites. It has been demonstrated that both mammalian PARP1 and PARP2 PARylate double-strand break termini in DNA oligonucleotide duplexes in vitro. Here, we show that mammalian PARP2 and PARP3 can PARylate and mono(ADP-ribosyl)ate (MARylate), respectively, 5'- and 3'-terminal phosphate residues at double- and single-strand break termini of a DNA molecule containing multiple strand breaks. PARP3-catalyzed DNA MARylation can be considered a new type of reversible post-replicative DNA modification. According to DNA substrate specificity of PARP3 and PARP2, we propose a putative mechanistic model of PARP-catalyzed strand break-oriented ADP-ribosylation of DNA termini. Notably, PARP-mediated DNA ADP-ribosylation can be more effective than PARPs' auto-ADP-ribosylation depending on the DNA substrates and reaction conditions used. Finally, we show an effective PARP3- or PARP2-catalyzed ADP-ribosylation of high-molecular-weight (∼3-kb) DNA molecules, PARP-mediated DNA PARylation in cell-free extracts and a persisting signal of anti-PAR antibodies in a serially purified genomic DNA from bleomycin-treated poly(ADP-ribose) glycohydrolase-depleted HeLa cells. These results suggest that certain types of complex DNA breaks can be effectively ADP-ribosylated by PARPs in cellular response to DNA damage.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN , ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , ADN/química , Aductos de ADN/metabolismo , Roturas del ADN de Doble Cadena , Células HeLa , Humanos , Fosfatos/metabolismo , Especificidad por Sustrato
12.
Chemphyschem ; 20(8): 1028-1035, 2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30848540

RESUMEN

Among cationic, anionic, and merocyanine polymethine dyes, the binding to detonation nanodiamond (DND) colloid particles in hydrosol occurs only for negatively charged dye species. This, in view of the positive ζ-potential of the DND used in this study, suggests the predominance of electrostatic interactions over other intermolecular forces in such systems. Indeed, after decorating the merocyanine and the cationic dye by one and two negatively charged sulfopropyl groups, respectively, so that the net charge of their colored species becomes negative, the compounds also demonstrate affinity to the DND particles. In all cases, the binding of dyes to DND is accompanied by a decrease in fluorescence intensity and a bathochromic shift of their absorption and fluorescence bands. A quantitative study of the dyes adsorption on the DND nanoparticles as performed using the Küster-Freundlich and Langmuir equations reveals some peculiarities of their attaching to the DND aggregates and allows estimating the specific area of the DND particles at a concentration of 0.0212 wt/vol %.

13.
Mol Cell ; 43(4): 649-62, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21855803

RESUMEN

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.


Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Proteína 2 Homóloga a MutS/fisiología , Estrés Oxidativo , Antígeno Nuclear de Célula en Proliferación/fisiología , Proteínas de Arabidopsis , Línea Celular , Cromatina/metabolismo , ADN Polimerasa beta/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Endopeptidasas/metabolismo , Humanos , Proteína 2 Homóloga a MutS/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteasas Ubiquitina-Específicas , Ubiquitinación , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
14.
J Phys Chem A ; 122(50): 9645-9652, 2018 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-30452263

RESUMEN

Absorption and fluorescence spectra of a vinylogous series of reversely solvatochromic merocyanines based on benzimidazole and malononitrile have been studied in frozen ethanol solutions at 77 K. It is found that they possess negative thermochromism-in contrast to both positively solvatochromic merocyanines and negatively solvatochromic symmetrical ionic polymethines-and even stronger negative thermofluorochromism. It has been deduced from the spectral data that at low temperature their electronic structure becomes more dipolar, deviating substantially from the virtual ideal polymethine in both the ground and the excited states. At that, owing probably to the high polarity and ordering of frozen ethanol, the dipolarity of the studied merocyanines increases with the polymethine chain lengthening-the tendency not observed for them in common solvents. The conclusions, based on the spectral data analysis, have been verified by the (TD)DFT-PCM simulations of the dyes within the four-level scheme of electronic transitions.

15.
Nucleic Acids Res ; 44(8): 3713-27, 2016 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-26843430

RESUMEN

Active DNA demethylation (ADDM) in mammals occurs via hydroxylation of 5-methylcytosine (5mC) by TET and/or deamination by AID/APOBEC family enzymes. The resulting 5mC derivatives are removed through the base excision repair (BER) pathway. At present, it is unclear how the cell manages to eliminate closely spaced 5mC residues whilst avoiding generation of toxic BER intermediates and whether alternative DNA repair pathways participate in ADDM. It has been shown that non-canonical DNA mismatch repair (ncMMR) can remove both alkylated and oxidized nucleotides from DNA. Here, a phagemid DNA containing oxidative base lesions and methylated sites are used to examine the involvement of various DNA repair pathways in ADDM in murine and human cell-free extracts. We demonstrate that, in addition to short-patch BER, 5-hydroxymethyluracil and uracil mispaired with guanine can be processed by ncMMR and long-patch BER with concomitant removal of distant 5mC residues. Furthermore, the presence of multiple mispairs in the same MMR nick/mismatch recognition region together with BER-mediated nick formation promotes proficient ncMMR resulting in the reactivation of an epigenetically silenced reporter gene in murine cells. These findings suggest cooperation between BER and ncMMR in the removal of multiple mismatches that might occur in mammalian cells during ADDM.


Asunto(s)
5-Metilcitosina/metabolismo , Reparación de la Incompatibilidad de ADN , Reparación del ADN , Animales , Línea Celular , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Proteína 2 Homóloga a MutS/genética , Pentoxil (Uracilo)/análogos & derivados , Pentoxil (Uracilo)/metabolismo , Regiones Promotoras Genéticas , Uracilo/metabolismo
16.
Nucleic Acids Res ; 44(19): 9279-9295, 2016 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-27471034

RESUMEN

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3'-cordycepin, 5'- and 3'-phosphate and also to 5'-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5'-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2'-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2',1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1' of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.


Asunto(s)
Roturas del ADN de Doble Cadena , ADN/genética , ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Catálisis , Aductos de ADN , Humanos , Hidrólisis , Ratones , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Especificidad por Sustrato
17.
J Biol Chem ; 290(23): 14338-49, 2015 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-25869130

RESUMEN

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.


Asunto(s)
ADN Bacteriano/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Secuencia de Bases , Reparación del ADN , ADN Bacteriano/química , Desoxirribonucleasa (Dímero de Pirimidina)/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica
18.
Biochim Biophys Acta ; 1850(6): 1297-309, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25766873

RESUMEN

BACKGROUND: The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2+ ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis. METHODS: The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. RESULTS: We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. CONCLUSIONS: We consider His-83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. GENERAL SIGNIFICANCE: Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents.


Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , ADN de Hongos/química , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Histidina , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Zinc/metabolismo
19.
Nucleic Acids Res ; 42(10): 6300-13, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24692658

RESUMEN

The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N(6)-ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.


Asunto(s)
Islas de CpG , Reparación del ADN , Mutación , Timina ADN Glicosilasa/metabolismo , Adenina/química , Animales , Disparidad de Par Base , Células Cultivadas , ADN/metabolismo , Daño del ADN , Humanos , Ratones , Oligonucleótidos/química , Polimorfismo de Nucleótido Simple , Timina/química , Timina/metabolismo
20.
Proc Natl Acad Sci U S A ; 110(33): E3071-80, 2013 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-23898172

RESUMEN

8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases--Escherichia coli Xth and human APE1--can remove 5'S cdA (S-cdA) at 3' termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3' end. To understand the structural basis of 3' repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2'-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3'-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3'→5' exonuclease function and its inhibition by 3'-terminal S-cdA residue. This structure was used as a template to model 3'-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3' repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3' DNA termini in E. coli and human cells, respectively.


Asunto(s)
Aductos de ADN/metabolismo , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido)/química , Proteínas de Escherichia coli/química , Exonucleasas/metabolismo , Modelos Moleculares , Conformación Proteica , Aductos de ADN/química , Reparación del ADN/genética , Electroforesis en Gel de Gradiente Desnaturalizante , Desoxiadenosinas/química , Desoxiadenosinas/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Escherichia coli , Humanos , Estructura Molecular , Oligonucleótidos/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Difracción de Rayos X , Levaduras
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