RESUMEN

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.

Asunto(s)

ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleótidos de Desoxiadenina/genética , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiguanina/genética , Desoxirribonucleótidos/genética , Flúor/química , Biología Sintética/métodos , Nucleótidos de Timina/genética , Transcripción Genética , Proteínas Virales/metabolismo , Nucleótidos de Desoxiguanina/química , Especificidad por Sustrato

RESUMEN

To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity.

Asunto(s)

Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxiuracil/metabolismo , Pirofosfatasas/metabolismo , Nucleótidos de Timina/metabolismo , Línea Celular , Proliferación Celular , Daño del ADN , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiuracil/genética , Técnicas de Inactivación de Genes , Inestabilidad Genómica , Humanos , Células MCF-7 , Mutación , Pirofosfatasas/genética , Nucleótidos de Timina/genética

RESUMEN

We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components of two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable to hydrolysis than standard dNTPs. Steady-state kinetics studies show that the dimers exhibit reasonably good efficiency with the Klenow fragment of DNA polymerase I, and we identify thermostable enzymes that process them efficiently at high temperature. Experiments show that the dAp5dT dimer successfully acts as a combination of dATP and dTTP in primer extension reactions, and the dGp5dC dimer as a combination of dGTP and dCTP. The two dimers in combination promote successful 4-base primer extension. The final byproduct of the reaction, triphosphate, is shown to be less inhibitory to primer extension than pyrophosphate, the canonical byproduct. Finally, we document PCR amplification of DNA with two dimeric nucleotides, and show that the dimers can promote amplification under extended conditions when PCR with normal dNTPs fails. These dimeric nucleotides represent a novel and simple approach for increasing stability of nucleotides and avoiding inhibition from pyrophosphate.

Asunto(s)

ADN Polimerasa I/genética , Replicación del ADN/genética , ADN/biosíntesis , Nucleótidos/genética , ADN/genética , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiguanina/genética , Cinética , Temperatura

RESUMEN

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.

Asunto(s)

Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleótidos de Timina/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Decitabina/química , Decitabina/metabolismo , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxiguanina/genética , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Microalgas/genética , Microorganismos Modificados Genéticamente , Tasa de Mutación , Péptido Hidrolasas/genética , Timidina Quinasa/genética , Timidilato Sintasa/genética , Nucleótidos de Timina/genética

RESUMEN

Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, a structural basis for D-stereoselectivity against L-dNTPs, enantiomers of natural nucleotides (D-dNTPs), by any DNA polymerase or reverse transcriptase has not been established due to lack of a ternary structure of a polymerase, DNA, and an incoming L-dNTP. Here, we report 2.10-2.25 Å ternary crystal structures of human DNA polymerase λ, DNA, and L-deoxycytidine 5'-triphosphate (L-dCTP), or the triphosphates of lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) with four ternary complexes per asymmetric unit. The structures of these 12 ternary complexes reveal that relative to D-deoxycytidine 5'-triphosphate (D-dCTP) in the canonical ternary structure of Polλ-DNA-D-dCTP, L-dCTP, (-)3TC-TP, and (-)FTC-TP all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide, two are similar and show that the L-nucleotide forms three Watson-Crick hydrogen bonds with the templating nucleotide dG and adopts a chair-like triphosphate conformation. In the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds, whereas the templating dG is anchored by a hydrogen bond with the side chain of a semiconserved residue Y505. Furthermore, the triphosphate of the L-nucleotide adopts an unprecedented N-shaped conformation. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for the formation of the abovementioned four complexes along proposed catalytic pathways for L-nucleotide incorporation and provide the structural basis for the D-stereoselectivity of a DNA polymerase.

Asunto(s)

ADN Polimerasa beta/química , Nucleótidos de Desoxicitosina/química , Sustitución de Aminoácidos , ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxicitosina/metabolismo , Humanos , Enlace de Hidrógeno , Mutación Missense , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato

RESUMEN

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.

Asunto(s)

Replicación del ADN , ADN Mitocondrial/genética , Nucleótidos de Desoxicitosina/metabolismo , Encefalomiopatías Mitocondriales/genética , Nucleótidos de Timina/metabolismo , Animales , Técnicas de Cultivo de Célula , Nucleótidos de Desoxicitosina/genética , Fibroblastos/citología , Humanos , Ratones , Mitocondrias Hepáticas/metabolismo , Encefalomiopatías Mitocondriales/metabolismo , Nucleótidos de Timina/genética

RESUMEN

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.

Asunto(s)

Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/genética , Guanosina/análogos & derivados , Dominio Catalítico , Cristalografía por Rayos X , Nucleótidos de Desoxicitosina/genética , Guanosina/genética , Humanos , Conformación de Ácido Nucleico , Estrés Oxidativo , Especificidad por Sustrato , ADN Polimerasa iota

RESUMEN

We studied usage of cytosine and guanine in 914 genes from completely sequenced genomes of five Simplex- and seven Varicelloviruses. In genes with total GC-content higher than 50% usage of cytosine is usually higher than usage of guanine (an average difference for genes with G+C higher than 70% reaches 4.0%). This difference is caused mostly by the elevated usage of cytosine in two-fold degenerated sites situated in third codon positions relatively to the usage of guanine in two-fold degenerated sites situated in third codon positions (an average difference for genes with G+C higher than 70% is equal to 28.2%). The usage of amino acids that are encoded by codons containing cytosine in two-fold degenerated sites situated in third codon positions (AA2TC) is much higher than the usage of amino acids encoded by codons containing guanine in two-fold degenerated sites situated in third codon positions (AA2AG). The usage of AA2AG declines much more steeply with the growth of GC-content than the usage of AA2TC. This effect is the consequence of the nature of genetic code and of the negative selection. In GC-rich genes the usage of cytosine in four-fold degenerated sites is only a little (but significantly) higher than the usage of guanine (in genes with G+C higher than 70% an average difference is equal to 4.3%). This difference may be caused by transcription-associated mutational pressure.

Asunto(s)

Composición de Base/genética , Codón/genética , Citosina/análisis , Genes Virales/genética , Guanina/análisis , Simplexvirus/genética , Varicellovirus/genética , Aminoácidos/genética , Disparidad de Par Base/genética , ADN de Cadena Simple/genética , ADN Viral/química , ADN Viral/genética , Nucleótidos de Desoxicitosina/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/genética , Código Genético/genética , Mutación Puntual/genética , Selección Genética/genética

RESUMEN

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.

Asunto(s)

Disparidad de Par Base/genética , Productos Finales de Glicación Avanzada/química , Productos Finales de Glicación Avanzada/genética , Guanosina/análogos & derivados , Mutágenos/síntesis química , Catálisis , Aductos de ADN/síntesis química , Aductos de ADN/genética , Aductos de ADN/metabolismo , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/genética , Desoxirribonucleótidos/síntesis química , Desoxirribonucleótidos/genética , Desoxirribonucleótidos/metabolismo , Glicosilación , Guanosina/síntesis química , Guanosina/genética , Guanosina/metabolismo , Humanos , Pruebas de Mutagenicidad , Mutágenos/metabolismo , Estereoisomerismo , Moldes Genéticos

RESUMEN

Global hypomethylation has long been recognized as a feature of the malignant epithelial component in human carcinomas. Here we show evidence for this same type of epigenetic alteration in cancer-associated stromal myofibroblasts. We used methylation-sensitive SNP array analysis (MSNP) to profile DNA methylation in early-passage cultures of stromal myofibroblasts isolated from human gastric cancers. The MSNP data indicated widespread hypomethylation in these cells, with rare focal gains of methylation, conclusions that were independently validated by bisulfite sequencing and by a methylation-sensitive cytosine incorporation assay. Immunohistochemistry with anti-5-methylcytosine (anti-5-methyl-C) in a series of gastrectomy specimens showed frequent loss of methylation in nuclei of both the malignant epithelial cells and alpha-smooth muscle actin (ASMA)-positive stromal myofibroblasts of both intestinal-type and diffuse carcinomas. We confirmed this phenomenon and established its onset at the stage of noninvasive dysplastic lesions by immunohistochemistry for anti-5-methyl-C in a transgenic mouse model of multistage gastric carcinogenesis. These findings indicate similar general classes of epigenetic alterations in carcinoma cells and their accompanying reactive stromal cells and add to accumulating evidence for biological differences between normal and cancer-associated myofibroblasts.

Asunto(s)

Metilación de ADN , ADN de Neoplasias/genética , Neoplasias Gástricas/genética , Animales , Inestabilidad Cromosómica , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/genética , ADN de Neoplasias/metabolismo , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxicitosina/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/patología , Humanos , Inmunohistoquímica , Ratones , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas/patología , Células del Estroma/patología

RESUMEN

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia/métodos , Nucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Carbocianinas/química , ADN/química , ADN/genética , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/genética , Fluoresceínas/química , Colorantes Fluorescentes/química , Frecuencia de los Genes , Genotipo , Nucleótidos/química , Reacción en Cadena de la Polimerasa/métodos , Receptor de Serotonina 5-HT2A/genética , Reproducibilidad de los Resultados

RESUMEN

Expression of the glucose transporter GLUT1 is high in proliferating and tumour cells. Conditions in which the transcriptional activity of GLUT1 promoter are maximal are characterized by the operation of activators such as Sp1 and inhibitors as Sp3. Here, we have identified an element at -67/-60 (C8 box) in the rat GLUT1 promoter to which nuclear factors bind in a proliferation-dependent manner. Competition studies with a series of mutated oligonucleotides suggest that these nuclear factors are different in the proliferative state and after confluence. The C8 element does not bind any of the known factors defined in databases. Mutation of the C8 sequence enhanced transcriptional activity of the GLUT1 promoter in 10T1/2 fibroblasts and in L6E9 myoblasts but not in myotubes. Furthermore, the functional activity of the C8 box is maximal in the presence of serum. In summary, we have identified a novel inhibitory element, C8 box, in the rat GLUT1 promoter that operates in a proliferation-dependent manner. In keeping with this view, serum enhances the inhibitory activity of the C8 box, suggesting that this is regulated by growth factors.

Asunto(s)

Proteínas de Transporte de Monosacáridos/genética , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Animales , Sitios de Unión/genética , División Celular/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Nucleótidos de Desoxicitosina/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1 , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Proteínas Nucleares/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección

RESUMEN

The intermediate produced from 5-methyl-2'-deoxycytidine ((5me)dCyd) by HNO2 and NO treatments was isolated and characterized. When 10mM (5me)dCyd was incubated with 100mM NaNO2 at pH 3.7 and 37 degrees C, a previously unidentified product was formed. The product was identified as a diazoate derivative of (5me)dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-5-methyl-2-oxopyrimidine-4-diazoate ((5me)dCyd-diazoate), on the bases of several measurements including LC/MS. The time course of the concentration change of the diazoate showed a characteristic profile of a reaction intermediate, and the steady state concentration was 2.3 microM (0.023% yield). When an aqueous solution of 10mM (5me)dCyd (10 mL) was bubbled by NO at 37 degrees C under aerobic conditions holding the pH around 7.4, the diazoate was also generated. The yield of the diazoate was 0.041 micromol (0.041% yield) at 20 mmol of NO absorption. At physiological pH and temperature (pH 7.4, 37 degrees C), the diazoate was converted to dThd exclusively with a first order rate constant k=9.1x10(-6) x s(-1) (t(1/2)=21 h). These results show that the diazoate is generated as a relatively stable intermediate in the reactions of (5me)dCyd with HNO2 and NO and further suggest that the diazoate can be formed in cellular DNA with biologically relevant doses of HNO2 and NO.

Asunto(s)

Compuestos Azo/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Desoxiguanosina/análogos & derivados , Óxido Nítrico/metabolismo , Ácido Nitroso/metabolismo , Compuestos Azo/química , ADN/efectos de los fármacos , ADN/genética , ADN/metabolismo , Desoxicitidina/química , Desoxicitidina/genética , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxicitosina/metabolismo , Desoxiguanosina/química , Desoxiguanosina/genética , Desoxiguanosina/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Estabilidad de Medicamentos , Cinética , Modelos Genéticos , Estructura Molecular , Mutación

RESUMEN

Nucleotide pool imbalances have been reported to affect the fidelity of DNA replication and repair in prokaryotic and eukaryotic cells. We have reported previously that the mutagen-hypersensitive thymidine kinase (TK)-deficient Friend erythroleukemia (FEL) cells (subclones 707BUF and 707BUE), have a more than sixfold increase in the dCTP:dTTP pool ratio when compared to that of wild-type, TK-positive (TK(+)) clone 707 cells. In this study we present the results of an investigation of the effect of the dCTP:dTTP pool imbalance on the accuracy of DNA replication within 707BUF cells. We examined the spontaneous mutation spectra occurring at the adenine phosphoribosyltransferase (aprt) locus within clone 707 (TK(+)) and 707BUF (TK(-)) FEL cells. Mutations recovered at the aprt locus in FEL cells comprised: base substitutions (43:73), frameshifts (14:13.5), and deletions (43:13.5) [clone 707 (TK(+)):707BUF (TK(-)), respectively, expressed as percentages]. A comparison of the mutation spectra obtained for the two cell lines did not reveal any significant increase in misincorporation of dCTP, the nucleotide in excess, in 707BUF (TK(-)) cells, during DNA replication synthesis. These data suggest that the dCTP:dTTP pool imbalance does not alter the fidelity of DNA replication synthesis in 707BUF (TK(-)) FEL cells. Rather, the predominance of GC --> AT transitions (53%) in the 707BUF (TK(-)) spectrum may reflect a reduced efficiency of repair by uracil DNA glycosylase of uracil residues within these cells.

Asunto(s)

Replicación del ADN/genética , Nucleótidos de Desoxicitosina/metabolismo , Leucemia Eritroblástica Aguda/genética , Nucleótidos de Timina/metabolismo , Adenina Fosforribosiltransferasa/genética , Animales , Células Clonales , Análisis Mutacional de ADN/métodos , Nucleótidos de Desoxicitosina/genética , Exones , Leucemia Eritroblástica Aguda/metabolismo , Ratones , Mutación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Timidina Quinasa/deficiencia , Timidina Quinasa/genética , Nucleótidos de Timina/genética , Células Tumorales Cultivadas

RESUMEN

The crystal structure of an alternating RNA octamer, r(guauaca)dC (RNA bases are in lower case while the only DNA base is in upper case), with two 3' overhang residues one of them a terminal deoxycytosine and the other a ribose adenine, has been determined at 2.2 A resolution. The refined structure has an Rwork 18.6% and Rfree 26.8%. There are two independent duplexes (molecules I and II) in the asymmetric unit cell, a = 24.95, b = 45.25 and c = 73.67 A, with space group P2(1)2(1)2(1). Instead of forming a blunt end duplex with two a+.c mispairs and six Watson-Crick base-pairs, the strands in the duplex slide towards the 3' direction forming a two-base overhang (radC) and a six Watson-Crick base-paired duplex. The duplexes are bent (molecule I, 20 degrees; molecule II, 25 degrees) and stack head-to-head to form a right-handed superhelix. The overhang residues are looped out and the penultimate adenines of the two residues at the top end (A15) are anti and at the bottom (A7) end are syn. The syn adenine bases form minor groove A*(G.C) base triples with C8-H...N2 hydrogen bonds. The anti adenine in molecule II also forms a triple and a different C2-H...N3 hydrogen bond, while the other anti adenine in molecule I does not, it stacks on the looped out overhang base dC. The 3' terminal deoxycytosines form two stacked hemiprotonated trans d(C.C)+ base-pairs and the pseudo dyad related molecules form four consecutive deoxyribose and ribose zipper hydrogen bonds in the minor groove.

Asunto(s)

Disparidad de Par Base/genética , Emparejamiento Base/genética , Nucleótidos de Desoxicitosina/genética , Ácidos Nucleicos Heterodúplex/genética , ARN/química , ARN/genética , Adenina/química , Adenina/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Nucleótidos de Desoxicitosina/química , Desoxirribosa/química , Desoxirribosa/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Oxígeno/metabolismo , Protones , Ribosa/química , Ribosa/metabolismo , Agua/metabolismo

RESUMEN

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Asunto(s)

Arabidopsis/genética , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiuracil/genética , Mitocondrias/genética , Sistemas de Lectura Abierta , Edición de ARN , Secuencia de Bases , Código Genético , Intrones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Planta/genética , ARN Ribosómico/genética , ARN de Transferencia/genética

RESUMEN

During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery. In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassed in vitro when they are short in length and are followed by homologous DNA downstream. Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery. The RuvA and RuvB proteins of Escherichia coli form an important part of this machinery. In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by the E. coli RecA protein. Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions. Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes. Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

Asunto(s)

Proteínas Bacterianas/metabolismo , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Ácidos Nucleicos Heterodúplex/química , Recombinación Genética/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Citidina Trifosfato/química , Citidina Trifosfato/genética , Citidina Trifosfato/metabolismo , Reparación del ADN/genética , ADN Circular/química , ADN Circular/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxicitosina/metabolismo , Proteínas de Escherichia coli , Cinética , Modelos Genéticos , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Rec A Recombinasas/química , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo

RESUMEN

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Asunto(s)

Yema de Huevo/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Pez Cebra/genética , Actinas/genética , Animales , Animales Modificados Genéticamente , Núcleo Celular/genética , Nucleótidos de Desoxicitosina/genética , Yema de Huevo/metabolismo , Embrión no Mamífero/ultraestructura , Femenino , Vectores Genéticos/química , Células Gigantes , Masculino , Microinyecciones , Radioisótopos de Fósforo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.

Asunto(s)

Replicación del ADN , Nucleótidos de Desoxicitosina/genética , Mutación , Animales , Composición de Base , Secuencia de Bases , Células Cultivadas , Cricetinae , Cricetulus , Reparación del ADN , Nucleótidos de Desoxicitosina/metabolismo , Femenino , Ovario

RESUMEN

In crosses between bacteriophages T2 and T4 most early genes of T2 are partially excluded from the progeny. Six genes of T4 affect the exclusion of six corresponding exclusion-sensitive sites in T2, each gene being specific for the exclusion of one site. Mutants of T4 in these genes have been isolated (ex mutants). The induction of the gene product (deoxycytidine triphosphate nucleotidohydrolase, dCTPase) of the strongly excluded T2 gene 56 was determined. The dCTPase was induced in the presence of the replication inhibitor oxolinic acid to prevent possible artefacts from unequal replicaton rates of T2 and T4. The rate of T2 dCTPase induction was 30% of the control in T2 X T4 infections in which all six exclusion-sensitive sites were excluded and was 57% in infections in which the sites except 56 were excluded. The dCTPase induction was 67% of the control with exclusion of the 56 site only and equalled the control when no T2 site was excluded. Inhibition of dCTPase induction under conditions of exclusion is partly due to exclusion of neighboring sites and partly due to exclusion of the 56 site.

Asunto(s)

Genes Virales , Monoéster Fosfórico Hidrolasas/biosíntesis , Fosfotransferasas (Aceptor del Grupo Fosfato) , Pirofosfatasas , Fagos T/genética , Nucleótidos de Desoxicitosina/biosíntesis , Nucleótidos de Desoxicitosina/genética , Inducción Enzimática , Cinética , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/biosíntesis , Fagos T/enzimología , Fagos T/crecimiento & desarrollo