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1.
Mutat Res ; 852: 503160, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265045

RESUMO

Professor Barbara Tudek received the Frits Sobels Award in 2019 from the European Environmental Mutagenesis and Genomics Society (EEMGS). This article presents her outstanding character and most important lines of research. The focus of her studies covered alkylative and oxidative damage to DNA bases, in particular mutagenic and carcinogenic properties of purines with an open imidazole ring and 8-oxo-7,8-dihydroguanine (8-oxoGua). They also included analysis of mutagenic properties and pathways for the repair of DNA adducts of lipid peroxidation (LPO) products arising in large quantities during inflammation. Professor Tudek did all of this in the hope of deciphering the mechanisms of DNA damage removal, in particular by the base excision repair (BER) pathway. Some lines of research aimed at discovering factors that can modulate the activity of DNA damage repair in hope to enhance existing anti-cancer therapies. The group's ongoing research aims at deciphering the resistance mechanisms of cancer cell lines acquired following prolonged exposure to photodynamic therapy (PDT) and the possibility of re-sensitizing cells to PDT in order to increase the application of this minimally invasive therapeutic method.


Assuntos
Carcinogênese/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Neoplasias/história , Fotoquimioterapia/história , Radiossensibilizantes/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Guanina/metabolismo , História do Século XX , História do Século XXI , Humanos , Peroxidação de Lipídeos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fotoquimioterapia/métodos
2.
BMC Bioinformatics ; 21(1): 40, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005130

RESUMO

BACKGROUND: Quadruplexes are specific structure motifs occurring, e.g., in telomeres and transcriptional regulatory regions. Recent discoveries confirmed their importance in biomedicine and led to an intensified examination of their properties. So far, the study of these motifs has focused mainly on the sequence and the tertiary structure, and concerned canonical structures only. Whereas, more and more non-canonical quadruplex motifs are being discovered. RESULTS: Here, we present ElTetrado, a software that identifies quadruplexes (composed of guanine- and other nucleobase-containing tetrads) in nucleic acid structures and classifies them according to the recently introduced ONZ taxonomy. The categorization is based on the secondary structure topology of quadruplexes and their component tetrads. It supports the analysis of canonical and non-canonical motifs. Besides the class recognition, ElTetrado prepares a dot-bracket and graphical representations of the secondary structure, which reflect the specificity of the quadruplex's structure topology. It is implemented as a freely available, standalone application, available at https://github.com/tzok/eltetrado. CONCLUSIONS: The proposed software tool allows to identify and classify tetrads and quadruplexes based on the topology of their secondary structures. It complements existing approaches focusing on the sequence and 3D structure.


Assuntos
Biologia Computacional/métodos , DNA/química , Quadruplex G , DNA/genética , Guanina/química , Guanina/metabolismo , Conformação de Ácido Nucleico , Software
3.
Life Sci ; 244: 117333, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962132

RESUMO

AIMS: Detect the antiarrhythmic effect of crotonoside (Cro). MAIN METHODS: We used whole-cell patch-clamp techniques to detect the effects of Cro on action potentials (APs) and transmembrane ion currents in isolated rabbit left ventricular myocytes. We also verified the effect of Cro on ventricular arrhythmias caused by aconitine in vivo. KEY FINDINGS: Cro reduced the maximum depolarization velocity (Vmax) of APs and shortened the action potential duration (APD) in a concentration-dependent manner, but it had no significant effect on the resting membrane potential (RMP) or action potential amplitude (APA). It also inhibited the peak sodium current (INa) and L-type calcium current (ICaL) in a concentration-dependent manner with half-maximal inhibitory concentrations (IC50) of 192 µmol/L and 159 µmol/L, respectively. However, Cro had no significant effects on the inward rectifier potassium current (IK1) or rapidly activating delayed rectifier potassium current (IKr). Sea anemone toxin II (ATX II) increased the late sodium current (INaL), but Cro abolished this effect. Moreover, Cro significantly abolished ATX II-induced early afterdepolarizations (EADs) and high extracellular Ca2+ concentration (3.6 mmol/L)-induced delayed afterdepolarizations (DADs). We also verified that Cro effectively delayed the onset time and reduced the incidence of ventricular arrhythmias caused by aconitine in vivo. SIGNIFICANCE: These results revealed that Cro effectively inhibits INa, INaL, and ICaL in ventricular myocytes. Cro has antiarrhythmic potential and thus deserves further study.


Assuntos
Guanina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , China , Feminino , Guanina/metabolismo , Ventrículos do Coração/metabolismo , Técnicas de Patch-Clamp/métodos , Coelhos , Sódio/metabolismo , Canais de Sódio/efeitos dos fármacos
4.
Chem Commun (Camb) ; 56(12): 1839-1842, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31950946

RESUMO

Oxidative damage of guanine to 8-oxoguanine triggers a partial and variable loss of G-quadruplex/hemin DNAzyme activity and provides clues to the mechanistic origins of DNAzyme deactivation, which originates from an interplay between decreased G-quadruplex stability, lower hemin affinity and a modification of the nature of hemin binding sites.


Assuntos
DNA Catalítico/metabolismo , DNA Catalítico/química , Quadruplex G , Guanina/química , Guanina/metabolismo , Estrutura Molecular , Oxirredução
5.
Phys Chem Chem Phys ; 22(5): 2999-3007, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31957771

RESUMO

Infrared multiple photon dissociation (IRMPD) spectroscopy has been used to probe the structures of the three protonated base-pair mismatches containing 9-ethylguanine (9eG) in the gas phase. Computational chemistry has been used to determine the relative energies and compute the infrared spectra of these complexes. By comparing the IRMPD spectra with the computed spectra, in all cases, it was possible to deduce that what was observed experimentally were the lowest energy computed structures. The protonated complex between 9eG and 1-methylthymine (1mT) is protonated at N7 of 9eG-the most basic site of all three bases in this study-and bound in a Hoogsteen type structure with two hydrogen bonds. The experimental IRMPD spectrum for the protonated complex between 9eG and 9-methyladenine (9mA) is described as arising from a combination of the two lowest energy structures, both which are protonated at N1 of adenine and each containing two hydrogen bonds with 9eG being the acceptor of both. The protonated dimer of 9eG is protonated at N7 with an N7-H+-N7 ionic hydrogen bond. It also contains an interaction between a C-H of protonated guanine and the O6 carbonyl of neutral guanine which is manifested in a slight red shift of that carbonyl stretch. The protonated 9eG/9mA structures have been previously identified by X-ray crystallography in RNA and are contained within the protein database.


Assuntos
Gases/química , Guanina/análogos & derivados , Espectrofotometria Infravermelho , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Pareamento Incorreto de Bases , Cristalografia por Raios X , Guanina/química , Guanina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Fótons , Timina/análogos & derivados , Timina/química , Timina/metabolismo
6.
Genes (Basel) ; 10(9)2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547242

RESUMO

In teleost, pigment in the skin and scales played important roles in various biological processes. Iridophores, one of the main pigment cells in teleost, could produce silver pigments to reflect light. However, the specific mechanism of the formation of silver pigments is still unclear. In our previous study, some transparent mutant individuals were found in the carp-goldfish nucleocytoplasmic hybrid (CyCa hybrid) population. In the present study, using transparent mutants (TM) and wild type (WT) of the CyCa hybrid as a model, firstly, microscopic observations showed that the silver pigments and melanin were both lost in the scales of transparent mutants compared to that in wild types. Secondly, genetic study demonstrated that the transparent trait in the CyCa hybrid was recessively inherent, and controlled by an allele in line with Mendelism. Thirdly, RNA-Seq analysis showed that differential expression genes (DEGs) between wild type and transparent mutants were mainly enriched in the metabolism of guanine, such as hydrolase, guanyl nucleotide binding, guanyl ribonucleotide binding, and GTPase activity. Among the DEGs, purine nucleoside phosphorylase 4a (pnp4a) and endothelin receptor B (ednrb) were more highly expressed in the wild type compared to the transparent mutant (p < 0.05). Finally, miRNA-Seq analysis showed that miRNA-146a and miR-153b were both more highly expressed in the transparent mutant compared to that in wild type (p < 0.05). Interaction analysis between miRNAs and mRNAs indicated that miRNA-146a was associated with six DEGs (MGAT5B, MFAP4, GP2, htt, Sema6b, Obscn) that might be involved in silver pigmentation.


Assuntos
Carpa Dourada/genética , Mutação , Pigmentação da Pele/genética , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Genes Recessivos , Guanina/metabolismo , Melaninas/genética , Melaninas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo
7.
PLoS Comput Biol ; 15(9): e1007383, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31539370

RESUMO

G-quadruplexes (G4) are secondary structures formed by guanine-rich nucleic acid sequences and shown to exist in living cells where they participate in regulation of gene expression and chromosome maintenance. G-quadruplexes with solvent-exposed guanine tetrads show the tendency to associate together through cofacial stacking, which may be important for packaging of G4-forming sequences and allows for the design of higher-order G4 DNA structures. To understand the molecular driving forces for G4 association, here, we study the binding interaction between two parallel-stranded G-quadruplexes using all-atom molecular dynamics simulations. The predicted dimerization free energies show that direct binding through the 5'-G-tetrads is the most preferred of all possible end-to-end stacking orientations, consistently with all available experimental data. Decomposition of dimerization enthalpies in combination with simulations at varying ionic strength further indicate that the observed orientational preferences arise from a fine balance between the electrostatic repulsion of the sugar-phosphate backbones and favorable counterion binding at the dimeric interface. We also demonstrate how these molecular-scale findings can be used to devise means of controlling G4 dimerization equilibrium, e.g., by altering salt concentration and using G4-targeted ligands.


Assuntos
DNA , Quadruplex G , Guanina , Sequência de Bases , Biologia Computacional , Simulação por Computador , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Dimerização , Guanina/química , Guanina/metabolismo , Simulação de Dinâmica Molecular , Termodinâmica
8.
Appl Microbiol Biotechnol ; 103(19): 8021-8033, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372707

RESUMO

8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a ß-elimination reaction. The enzyme displays an optimal temperature, ca.85-95 °C, and an optimal pH, ca.7.0-8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Análise Mutacional de DNA , Guanina/análogos & derivados , Thermococcus/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Guanina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Temperatura
9.
Nat Struct Mol Biol ; 26(8): 695-703, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31332353

RESUMO

UV-DDB, a key protein in human global nucleotide excision repair (NER), binds avidly to abasic sites and 8-oxo-guanine (8-oxoG), suggesting a noncanonical role in base excision repair (BER). We investigated whether UV-DDB can stimulate BER for these two common forms of DNA damage, 8-oxoG and abasic sites, which are repaired by 8-oxoguanine glycosylase (OGG1) and apurinic/apyrimidinic endonuclease (APE1), respectively. UV-DDB increased both OGG1 and APE1 strand cleavage and stimulated subsequent DNA polymerase ß-gap filling activity by 30-fold. Single-molecule real-time imaging revealed that UV-DDB forms transient complexes with OGG1 or APE1, facilitating their dissociation from DNA. Furthermore, UV-DDB moves to sites of 8-oxoG repair in cells, and UV-DDB depletion sensitizes cells to oxidative DNA damage. We propose that UV-DDB is a general sensor of DNA damage in both NER and BER pathways, facilitating damage recognition in the context of chromatin.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Linhagem Celular , Dano ao DNA , DNA Glicosilases/química , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Dímeros de Pirimidina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula , Especificidade por Substrato , Xeroderma Pigmentoso/patologia
10.
Nucleic Acids Res ; 47(14): 7276-7293, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31318975

RESUMO

Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.


Assuntos
DNA de Cadeia Simples/química , DNA/química , Quadruplex G , Guanina/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Guanina/metabolismo , Humanos , Cinética
11.
Nucleic Acids Res ; 47(15): 8154-8162, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31276580

RESUMO

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.


Assuntos
Adenina/metabolismo , DNA Catalítico/metabolismo , Guanina/metabolismo , Sódio/metabolismo , Algoritmos , Sequência de Bases , Sítios de Ligação/genética , Biocatálise , DNA Catalítico/química , DNA Catalítico/genética , Cinética , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , RNA/genética , RNA/metabolismo , Especificidade por Substrato
12.
Nucleic Acids Res ; 47(13): 6618-6631, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31173143

RESUMO

Riboswitches can regulate gene expression by direct and specific interactions with ligands and have recently attracted interest as potential drug targets for antibacterial. In this work, molecular dynamics (MD) simulations, free energy perturbation (FEP) and molecular mechanics generalized Born surface area (MM-GBSA) methods were integrated to probe the effect of mutations on the binding of ligands to guanine riboswitch (GR). The results not only show that binding free energies predicted by FEP and MM-GBSA obtain an excellent correlation, but also indicate that mutations involved in the current study can strengthen the binding affinity of ligands GR. Residue-based free energy decomposition was applied to compute ligand-nucleotide interactions and the results suggest that mutations highly affect interactions of ligands with key nucleotides U22, U51 and C74. Dynamics analyses based on MD trajectories indicate that mutations not only regulate the structural flexibility but also change the internal motion modes of GR, especially for the structures J12, J23 and J31, which implies that the aptamer domain activity of GR is extremely plastic and thus readily tunable by nucleotide mutations. This study is expected to provide useful molecular basis and dynamics information for the understanding of the function of GR and possibility as potential drug targets for antibacterial.


Assuntos
2-Aminopurina/análogos & derivados , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Hipoxantina/metabolismo , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Riboswitch/genética , 2-Aminopurina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Guanina/metabolismo , Ligantes , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Termodinâmica
13.
Nucleic Acids Res ; 47(10): 5049-5060, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916339

RESUMO

Oxidation of the guanine (G) heterocycle to 8-oxo-7,8-dihydroguanine (OG) in mammalian gene promoters was demonstrated to induce transcription. Potential G-quadruplex forming sequences (PQSs) in promoters have a high density of G nucleotides rendering them highly susceptible to oxidation and possible gene activation. The VEGF PQS with OG or an abasic site were synthesized at key locations in the SV40 or HSV-TK model promoters to determine the location dependency in the gene expression profile in human cells. The PQS location with respect to the transcription start site (TSS) and strand of occupancy (coding versus non-coding strand) are key parameters that determine the magnitude and direction in which gene expression changes with the chemically modified VEGF PQS. The greatest impact observed for OG or F in the PQS context in these promoters was within ∼200 bp of the TSS. Established PQSs found to occur naturally in a similar location relative to the TSS for possible oxidation-induced gene activation include c-MYC, KRAS, c-KIT, HIF-1α, PDGF-A and hTERT. The studies provide experimental constraints that were used to probe bioinformatic data regarding PQSs in the human genome for those that have the possibility to be redox switches for gene regulation.


Assuntos
Quadruplex G , Regulação da Expressão Gênica , Guanina/análogos & derivados , Guanina/química , Estresse Oxidativo , Regiões Promotoras Genéticas/genética , Sequência de Bases , Linhagem Celular Tumoral , Biologia Computacional/métodos , Reparo do DNA , Genoma Humano/genética , Guanina/metabolismo , Células HeLa , Células Hep G2 , Humanos , Oxirredução , Sítio de Iniciação de Transcrição
14.
Nucleic Acids Res ; 47(7): 3711-3727, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30715423

RESUMO

In eukaryotes, the wobble position of tRNA with a GUN anticodon is modified to the 7-deaza-guanosine derivative queuosine (Q34), but the original source of Q is bacterial, since Q is synthesized by eubacteria and salvaged by eukaryotes for incorporation into tRNA. Q34 modification stimulates Dnmt2/Pmt1-dependent C38 methylation (m5C38) in the tRNAAsp anticodon loop in Schizosaccharomyces pombe. Here, we show by ribosome profiling in S. pombe that Q modification enhances the translational speed of the C-ending codons for aspartate (GAC) and histidine (CAC) and reduces that of U-ending codons for asparagine (AAU) and tyrosine (UAU), thus equilibrating the genome-wide translation of synonymous Q codons. Furthermore, Q prevents translation errors by suppressing second-position misreading of the glycine codon GGC, but not of wobble misreading. The absence of Q causes reduced translation of mRNAs involved in mitochondrial functions, and accordingly, lack of Q modification causes a mitochondrial defect in S. pombe. We also show that Q-dependent stimulation of Dnmt2 is conserved in mice. Our findings reveal a direct mechanism for the regulation of translational speed and fidelity in eukaryotes by a nutrient originating from bacteria.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Micronutrientes/genética , Biossíntese de Proteínas/genética , Proteínas de Schizosaccharomyces pombe/genética , Animais , Anticódon/genética , Asparagina/genética , DNA Mitocondrial/genética , Eucariotos/genética , Guanina/análogos & derivados , Guanina/metabolismo , Metilação , Camundongos , RNA de Transferência/genética , Ribossomos/genética , Schizosaccharomyces/genética , Tirosina/genética
15.
BMC Res Notes ; 12(1): 92, 2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30777129

RESUMO

OBJECTIVES: Reactive oxygen species (ROS) oxidize guanine residues in DNA to form 7,8-dihydro-oxo-2'-deoxyguanosine (8oxoG) lesions in the genome. Human 8-oxoguanine glycosylase-1 (hOGG1) recognizes and excises this highly mutagenic species when it is base-paired opposite a cytosine. We sought to characterize biochemically several hOGG1 variants that have been found in cancer tissues and cell lines, reasoning that if these variants have reduced repair capabilities, they could lead to an increased chance of mutagenesis and carcinogenesis. RESULTS: We have over-expressed and purified the R46Q, A85S, R154H, and S232T hOGG1 variants and have investigated their repair efficiency and thermostability. The hOGG1 variants showed only minor perturbations in the kinetics of 8oxoG excision relative to wild-type hOGG1. Thermal denaturation monitored by circular dichroism revealed that R46Q hOGG1 had a significantly lower Tm (36.6 °C) compared to the other hOGG1 variants (40.9 °C to 43.2 °C). Prolonged pre-incubation at 37 °C prior to the glycosylase assay dramatically reduces the excision activity of R46Q hOGG1, has a modest effect on wild-type hOGG1, and a negligible effect on A85S, R154H, and S232T hOGG1. The observed thermolability of hOGG1 variants was mostly alleviated by co-incubation with stoichiometric amounts of competitor DNA.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Guanina/análogos & derivados , Desnaturação de Ácido Nucleico , DNA Glicosilases/genética , Reparo do DNA/genética , Guanina/metabolismo , Humanos , Mutação
17.
ACS Sens ; 4(2): 479-487, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30657307

RESUMO

Purine detection in the brain with fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFME) has become increasingly popular over the past decade; despite the growing interest, an in-depth analysis of how purines interact with the CFME at fast-scan rates has not been investigated. Here, we show how the functional group type and placement in the purine ring modulate sensitivity, electron transfer kinetics, and adsorption on the carbon-fiber surface. Similar investigations of catecholamine interaction at CFME with FSCV have informed the development of novel catecholamine-based sensors and is needed for purine-based sensors. We tested purine bases with either amino, carbonyl, or both functional groups substituted at different positions on the ring and an unsubstituted purine. Unsubstituted purine showed very little to no interaction with the electrode surface, indicating that functional groups are important for interaction at the CFME. Purine nucleosides and nucleotides, like adenosine, guanosine, and adenosine triphosphate, are most often probed using FSCV due to their rich extracellular signaling modalities in the brain. Because of this, the extent to which the ribose and triphosphate groups affect the purine-CFME interaction was also evaluated. Amino functional groups facilitated the interaction of purine analogues with CFME more than carbonyl groups, permitting strong adsorption and high surface coverage. Ribose and triphosphate groups decreased the oxidative current and slowed the interaction at the electrode which is likely due to steric effects and electrostatic repulsion. This work provides insight into the factors that affect purine-CFME interaction and conditions to consider when developing purine-targeted sensors for FSCV.


Assuntos
Fibra de Carbono/química , Eletroquímica/instrumentação , Purinas/química , Adenina/metabolismo , Adsorção , Encéfalo/metabolismo , Difusão , Guanina/metabolismo , Microeletrodos , Oxirredução , Purinas/metabolismo
18.
ACS Chem Biol ; 14(2): 214-222, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30645109

RESUMO

Carboxymethylation of DNA, including the formation of the DNA adduct O6-carboxymethylguanine ( O6-CMG), is associated with lifestyle factors, such as diet. It can impede replicative polymerases (Pols) and lead to replication fork stalling, or an alternative means for replication to proceed by translesion DNA synthesis (TLS). TLS requires specialized DNA Pols characterized by open and preformed active sites capable of preferential bypass of alkylated DNA adducts but that have high error rates, leading to mutations. Human TLS Pols can bypass O6-CMG with varying degrees of accuracy, but it is not known how the chemical structure of the O6-CMG adduct influences polymerase proficiency or fidelity. To better understand how adduct structure determines dNTP selection at lesion sites, we prepared DNA templates with a series of O6-CMG structural analogs and compared the primer extension patterns of Y- and X-family Pols in response to these modifications. The results indicate that the structure of the DNA adduct had a striking effect on dNTP selection by Pol κ and that an increased steric size influences the fidelity of Pol η, whereas Pol ι and ß function were only marginally affected. To test the hypothesis that specific hydrogen bonding interactions between the templating base and the incoming dNTP are a basis of this selection, we modeled the structural analogs with incoming dNTP in the Pol κ active site. These data indicate that the base pairing geometry and stabilization by a dense hydrogen bonding network are important molecular features for dNTP incorporation, providing a basis for understanding error-free bypass of O6-CMG by Pol κ.


Assuntos
Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Adutos de DNA/metabolismo , Guanina/química , Guanina/metabolismo , Humanos , Cinética
19.
Chem Res Toxicol ; 32(4): 753-761, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30688445

RESUMO

The interchange between different repair mechanisms in human cells has long been a subject of interest. Here, we provide a direct demonstration that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) embedded in double-stranded DNA are substrates of both base excision repair (BER) and nucleotide excision repair (NER) mechanisms in intact human cells. Site-specifically modified, 32P-internally labeled double-stranded DNA substrates were transfected into fibroblasts or HeLa cells, and the BER and/or NER mono- and dual incision products were quantitatively recovered after 2-8 h incubation periods and lysis of the cells. DNA duplexes bearing single benzo[ a]pyrene-derived guanine adduct were employed as positive controls of NER. The NER activities, but not the BER activities, were abolished in XPA-/- cells, while the BER yields were strongly reduced in NEIL1-/- cells. Co-transfecting different concentrations of analogous DNA sequences bearing the BER substrates 5-hydroxyuracil diminish the BER yields of Sp lesions and enhance the yields of NER products. These results are consistent with a model based on the local availability of BER and NER factors in human cells and their competitive binding to the same Sp or Gh BER/NER substrates.


Assuntos
Guanina/metabolismo , Células Cultivadas , Reparo do DNA , Fibroblastos/metabolismo , Guanina/química , Células HeLa , Humanos , Cinética , Estrutura Molecular , Oxirredução
20.
Chem Res Toxicol ; 32(3): 437-446, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30604962

RESUMO

Because of its low redox potential, guanine (G) is the most frequent site of oxidation in the genome. Metabolic processes generate reactive oxygen species (ROS) that can oxidize G to yield 8-oxo-7,8-dihydroguanine (OG) as a key two-electron oxidation product. In a genome, G-rich sites including many gene promoters are sensitive to oxidative modification, and some of these regions have the propensity to form G-quadruplexes (G4s). Recently, OG formation in G-rich gene promoters was demonstrated to regulate mRNA expression via the base excision repair (BER) pathway. The proliferating cell nuclear antigen ( PCNA) gene was previously found to be activated by metabolic ROS, and the gene has a five G-track potential G4 in the coding strand of its promoter. Herein, we demonstrated the ability for four G runs of the PCNA promoter sequence to adopt a parallel-stranded G4. Next, we identified G nucleotides in the PCNA G4 sequence sensitive to oxidative modification. The G oxidation product OG and its initial BER product, an abasic site, were synthetically incorporated into the four- and five-track PCNA sequences at the sensitive sites followed by interrogation of G4 folding by five methods. We found the modifications impacted the G4 folds with positional dependency. Additionally, the fifth G track maintained the stability of the modified G4s by extrusion of the oxidatively modified G run. Finally, we synthetically inserted a portion of the promoter into a reporter plasmid with OG at select oxidation-prone positions to monitor expression in human glioblastoma cells. Our results demonstrate that OG formation in the context of the PCNA G4 can lead to increased gene expression consistent with the previous studies identifying that metabolic ROS activates transcription of the gene. This study provides another example of a G4 with the potential to serve as a regulatory agent for gene expression upon G oxidation.


Assuntos
Quadruplex G , Antígeno Nuclear de Célula em Proliferação/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética , Glioblastoma , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
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