Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.342
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
DNA Cell Biol ; 39(1): 50-56, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31750734

RESUMO

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.


Assuntos
DNA Polimerase III/genética , Desoxirribonucleotídeos/metabolismo , Fibroblastos/metabolismo , Mutação , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/metabolismo , Surdez/genética , Surdez/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Lipodistrofia/genética , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Interferência de RNA , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Síndrome
2.
Postepy Biochem ; 65(2): 143-152, 2019 06 06.
Artigo em Polonês | MEDLINE | ID: mdl-31642653

RESUMO

High replication fidelity, understood as the DNA polymerases' ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme's catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , DNA/metabolismo , Instabilidade Genômica , Ribonucleotídeos/metabolismo , DNA/genética , Replicação do DNA , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Ribonucleotídeos/química
3.
Chin J Nat Med ; 17(8): 608-615, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472898

RESUMO

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.


Assuntos
Adenocarcinoma de Pulmão/patologia , Ciclo Celular/efeitos dos fármacos , Desoxirribonucleotídeos/metabolismo , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Resveratrol/farmacologia , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hidroxiureia/farmacologia , Neoplasias Pulmonares/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
4.
Anal Biochem ; 568: 65-72, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605633

RESUMO

Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.


Assuntos
Desoxirribonucleotídeos/análise , Técnicas de Diluição do Indicador , Espectrometria de Massas , Isótopos de Carbono , Células Cultivadas , Cromatografia Líquida , Desoxirribonucleotídeos/metabolismo , Humanos , Marcação por Isótopo , Isótopos de Nitrogênio
5.
J Mol Biol ; 430(24): 4908-4924, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30342933

RESUMO

Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a relatively high frequency in the genome of wild-type Thermococcales cells, and this frequency significantly increases upon deletion of RNase HII, the major enzyme responsible for the removal of RNA from DNA. Because ribonucleotides remain in genomic DNA, we then analyzed the effects on polymerization activities by the three DNA Pols. Depending on the identity of the base and the sequence context, all three DNA Pols bypass rNMP-containing DNA templates with variable efficiency and nucleotide (mis)incorporation ability. Unexpectedly, we found that PolD correctly base-paired a single ribonucleotide opposite rNMP-containing DNA templates. An evolutionary scenario is discussed concerning rNMP incorporation into DNA and genome stability.


Assuntos
DNA Arqueal/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Ribonucleotídeos/metabolismo , Thermococcales/genética , Proteínas Arqueais/metabolismo , Replicação do DNA , Desoxirribonucleotídeos/metabolismo , Instabilidade Genômica , Thermococcales/enzimologia
6.
Chembiochem ; 19(22): 2410-2420, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30204290

RESUMO

Deoxyxylonucleic acid (dxNA) is a synthetic polymer that might have potential for heredity and evolution. Because of dxNA's unusual backbone geometry, sequence information stored in it is presumed to be inaccessible to natural nucleic acids or proteins. Despite a large structural similarity with natural nucleotides, incorporation of 2'-deoxyxylonucleotides (dxNTs) through the action of polymerases is limited. We present the identification of a mutant of the DNA polymerase Therminator with increased tolerance to deoxyxylose-induced backbone distortions. Whereas the original polymerase stops after incorporation of two consecutive dxNTs, the mutant is able to catalyse the extension of incorporated dxNTs with 2'-deoxyribonucleotides (dNTs) and the incorporation of up to four dxNTs alternates with dNTs, thereby translocating a highly distorted double helix throughout the entire polymerase. A single His-to-Arg substitution very close to the catalytic site residues is held to be responsible for interaction with the primer phosphate groups and for stabilizing nucleotide sugar-induced distortions during incorporation and translocation.


Assuntos
Domínio Catalítico/genética , Primers do DNA/metabolismo , DNA Polimerase Dirigida por DNA , Desoxirribonucleotídeos/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação , Biologia Sintética
7.
ACS Synth Biol ; 7(6): 1565-1572, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29746092

RESUMO

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.


Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleotídeos de Timina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Decitabina/química , Decitabina/metabolismo , Nucleotídeos de Desoxicitosina/genética , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/genética , Nucleotídeos de Desoxiguanina/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Microalgas/genética , Microrganismos Geneticamente Modificados , Taxa de Mutação , Peptídeo Hidrolases/genética , Timidina Quinase/genética , Timidilato Sintase/genética , Nucleotídeos de Timina/genética
8.
Nucleic Acids Res ; 46(13): e78, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29718339

RESUMO

DNA polymerase fidelity is affected by both intrinsic properties and environmental conditions. Current strategies for measuring DNA polymerase error rate in vitro are constrained by low error subtype sensitivity, poor scalability, and lack of flexibility in types of sequence contexts that can be tested. We have developed the Magnification via Nucleotide Imbalance Fidelity (MagNIFi) assay, a scalable next-generation sequencing assay that uses a biased deoxynucleotide pool to quantitatively shift error rates into a range where errors are frequent and hence measurement is robust, while still allowing for accurate mapping to error rates under typical conditions. This assay is compatible with a wide range of fidelity-modulating conditions, and enables high-throughput analysis of sequence context effects on base substitution and single nucleotide deletion fidelity using a built-in template library. We validate this assay by comparing to previously established fidelity metrics, and use it to investigate neighboring sequence-mediated effects on fidelity for several DNA polymerases. Through these demonstrations, we establish the MagNIFi assay for robust, high-throughput analysis of DNA polymerase fidelity.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Desoxirribonucleotídeos/metabolismo
9.
Proc Natl Acad Sci U S A ; 115(5): 980-985, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29339523

RESUMO

The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the KMs of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Bactérias/genética , Replicação do DNA , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/genética , Desoxirribonucleotídeos/metabolismo , Cinética , Especificidade por Substrato , Taq Polimerase/genética , Taq Polimerase/metabolismo
10.
FEBS J ; 285(9): 1563-1571, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29288533

RESUMO

The term hypoxia refers to any condition where insufficient oxygen is available and therefore encompasses a range of actual oxygen concentrations. The regions of tumours adjacent to necrotic areas are at almost anoxic levels and are known to be extremely therapy resistant (radiobiological hypoxia). The biological response to radiobiological hypoxia includes the rapid accumulation of replication stress and subsequent DNA damage response, including both ATR- and ATM-mediated signalling, despite the absence of detectable DNA damage. The causes and consequences of hypoxia-induced replication stress will be discussed.


Assuntos
Hipóxia Celular/fisiologia , Replicação do DNA , Animais , Proteínas de Ciclo Celular/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Desoxirribonucleotídeos/metabolismo , Humanos , Neoplasias/genética , Oxigênio/farmacologia , Ribonucleotídeo Redutases/metabolismo , Estresse Fisiológico/genética , Microambiente Tumoral
11.
Science ; 358(6364): 797-802, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29123070

RESUMO

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/metabolismo , Adaptação Biológica , Cromatina/metabolismo , Desoxirribonucleotídeos/metabolismo , Humanos , Redes e Vias Metabólicas , Oxirredução , Transdução de Sinais
12.
J Exp Bot ; 68(21-22): 5773-5786, 2017 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-29186482

RESUMO

Deoxycytidine monophosphate deaminase (dCMP deaminase, DCD) is crucial to the production of dTTP needed for DNA replication and damage repair. However, the effect of DCD deficiency and its molecular mechanism are poorly understood in plants. Here, we isolated and characterized a rice albinic leaf and growth retardation (alr) mutant that is manifested by albinic leaves, dwarf stature and necrotic lesions. Map-based cloning and complementation revealed that ALR encodes a DCD protein. OsDCD was expressed ubiquitously in all tissues. Enzyme activity assays showed that OsDCD catalyses conversion of dCMP to dUMP, and the ΔDCD protein in the alr mutant is a loss-of-function protein that lacks binding ability. We report that alr plants have typical DCD-mediated imbalanced dNTP pools with decreased dTTP; exogenous dTTP recovers the wild-type phenotype. A comet assay and Trypan Blue staining showed that OsDCD deficiency causes accumulation of DNA damage in the alr mutant, sometimes leading to cell apoptosis. Moreover, OsDCD deficiency triggered cell cycle checkpoints and arrested cell progression at the G1/S-phase. The expression of nuclear and plastid genome replication genes was down-regulated under decreased dTTP, and together with decreased cell proliferation and defective chloroplast development in the alr mutant this demonstrated the molecular and physiological roles of DCD-mediated dNTP pool balance in plant development.


Assuntos
Ciclo Celular , DCMP Desaminase/genética , Reparo do DNA , Desoxirribonucleotídeos/metabolismo , Regulação da Expressão Gênica , Oryza/genética , Proteínas de Plantas/genética , DCMP Desaminase/metabolismo , Mutação , Oryza/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo
13.
Nucleic Acids Res ; 45(22): 12954-12962, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29165701

RESUMO

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex.


Assuntos
Algoritmos , DNA Viral/química , Vírus da Leucemia Murina/enzimologia , Modelos Químicos , RNA Viral/química , DNA Polimerase Dirigida por RNA/química , DNA Viral/genética , DNA Viral/metabolismo , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Cinética , Vírus da Leucemia Murina/genética , Pinças Ópticas , Polimerização , RNA Viral/genética , RNA Viral/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Moldes Genéticos , Termodinâmica
14.
Nucleic Acids Res ; 45(17): 10143-10155, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28973469

RESUMO

RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3' of such 'slippage-stimulatory' structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem-loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited.


Assuntos
DNA Complementar/genética , Mutação INDEL , Conformação de Ácido Nucleico , DNA Polimerase Dirigida por RNA/metabolismo , RNA/genética , Retroelementos/genética , Moldes Genéticos , Composição de Bases , Sequência de Bases , Domínio Catalítico , Desoxirribonucleotídeos/metabolismo , Proteínas de Drosophila/metabolismo , Indicadores e Reagentes , RNA/química , Retroviridae/enzimologia , Relação Estrutura-Atividade
15.
Nucleic Acids Res ; 45(15): 9138-9148, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911097

RESUMO

While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase µ (Pol µ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol µ with a ribonucleotide bound at the active site. These structures reveal that Pol µ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol µ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol µ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/química , DNA/química , Desoxirribonucleotídeos/química , Ribonucleotídeos/química , Motivos de Aminoácidos , Sequência de Bases , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleotídeos/metabolismo , Especificidade por Substrato , Termodinâmica
16.
Biochemistry ; 56(40): 5449-5456, 2017 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-28862868

RESUMO

The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.


Assuntos
DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Mutação , Neoplasias/genética , DNA Polimerase beta/química , Cinética , Modelos Moleculares , Neoplasias/enzimologia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Nucleotídeos de Timina/metabolismo
17.
DNA Repair (Amst) ; 57: 171-178, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28822913

RESUMO

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention.


Assuntos
Sobrevivência Celular , Dano ao DNA , Reparo do DNA , DNA/metabolismo , Polifosfatos/metabolismo , Desoxirribonucleotídeos/metabolismo , Células HEK293 , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia
18.
Bioconjug Chem ; 28(8): 2125-2134, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28686433

RESUMO

DNA polymerases are known to recognize preferably d-nucleotides over l-nucleotides during DNA synthesis. Here, we report that several general DNA polymerases catalyze polymerization reactions of nucleotides directed by the DNA template containing an l-thymidine (l-T). The results display that the 5'-3' primer extension of natural nucleotides get to the end at chiral modification site with Taq and Phanta Max DNA polymerases, but the primer extension proceeds to the end of the template catalyzed by Deep Vent (exo-), Vent (exo-), and Therminator DNA polymerases. Furthermore, templating l-nucleoside displays a lag in the deoxyribonucleotide triphosphate (dNTP) incorporation rates relative to natural template by kinetics analysis, and polymerase chain reactions were inhibited with the DNA template containing two or three consecutive l-Ts. Most interestingly, no single base mutation or mismatch mixture corresponding to the location of l-T in the template was found, which is physiologically significant because they provide a theoretical basis on the involvement of DNA polymerase in the effective repair of l-T that may lead to cytotoxicity.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Timidina/metabolismo , Pareamento de Bases , Sequência de Bases , DNA/biossíntese , DNA/genética , Replicação do DNA , Cinética , Estereoisomerismo
19.
Nat Rev Genet ; 18(9): 535-550, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28714480

RESUMO

The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.


Assuntos
Dano ao DNA , Replicação do DNA , Animais , Ciclo Celular , Células/metabolismo , Desoxirribonucleotídeos/metabolismo , Humanos , Conformação de Ácido Nucleico , Transcrição Genética
20.
Dev Cell ; 42(3): 301-308.e3, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28735680

RESUMO

Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. We examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized "on the go." The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis.


Assuntos
Desoxirribonucleotídeos/metabolismo , Drosophila/metabolismo , Retroalimentação Fisiológica , Animais , Desoxirribonucleotídeos/biossíntese , Drosophila/embriologia , Drosophila/genética , Desenvolvimento Embrionário
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA