RESUMEN
BACKGROUND: Colorectal cancer is the second cause of death by cancer around the world. Sporadic colorectal cancer is the most frequent (75%), and it is produced by the interaction of environmental, epigenetic, and genetic factors. The accumulation of single-nucleotide variants in genes associated with cell proliferation, DNA repair, and/or apoptosis could confer a risk to cancer. The aim of this study was to analyze the gene-gene interactions among CCND2 (rs3217901), CDKN1A (rs1059234 and rs1801270), and POLD3 (rs3824999) variants in Mexican patients with colorectal cancer. METHODS: We collected peripheral blood samples from 185 patients with sporadic colorectal cancer before treatment and from 185 unrelated blood donors as the reference group; all participants signed an informed consent form. DNA extraction was performed by Miller and Cetyltrimethylammonium bromide (CTAB)/ Dodecyltrimethylammonium bromide (DTAB) methods. Polymerase chain reaction- restriction fragment length polymorphism followed by polyacrylamide gel electrophoresis stained with AgNO3 methods were used to identify the variants rs3217901, rs1059234, rs1801270, and rs3824999. Odds ratio and single-nucleotide variant interaction were determined by single-locus analysis and Multifactorial Dimensionality Reduction software, respectively. RESULTS: No association was found for CCND2 and CDKN1A variants; yet, a significant association for the GG genotype, G allele, and recessive and additive models for the POLD3 variant was observed (P < .05). The single-nucleotide variant-single-nucleotide variant interaction revealed the combination rs1059234, rs3217901, and rs3824999 as the best model and the comparison showed an increased risk (P < .05). CONCLUSION: Single-locus and gene-gene interaction analyses disclosed that both the rs3824999 (POLD3) variant and the combination of rs3217901 (CCND2), rs1059234 (CDKN1A), and rs3824999 (POLD3) genotypes increase the risk for colorectal cancer in Mexican population.
Asunto(s)
Neoplasias Colorrectales , Polimorfismo de Nucleótido Simple , Estudios de Casos y Controles , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/genética , Ciclina D2/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ADN Polimerasa III , Genotipo , Humanos , México , NucleótidosRESUMEN
In the present study, molecular characterization of Fasciola flukes from Spain was performed to reveal the relation with the previously reported Peruvian F. hepatica population. The nuclear DNA markers, phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold), were used for species identification of Fasciola flukes. A total of 196 Fasciola flukes were identified as F. hepatica by pepck and pold, and 26 haplotypes were detected in mitochondrial NADH dehydrogenase subunit 1 (nad1). Only one of them was previously found in Spanish samples; which indicates the existence of high genetic diversity and population structure in F. hepatica from Spain. Three haplotypes were identical to those from Peruvian F. hepatica. The pairwise fixation index value confirmed a relatively close relationship between the Spanish and Peruvian F. hepatica samples. The Spanish samples showed clearly higher genetic variability than the Peruvian population. These results are discussed in relation with the hypothesis of the introduction of the parasite in America from Europe and recent evidence of pre-Hispanic F. hepatica from Argentina revealed by ancient DNA.
Asunto(s)
Enfermedades de los Bovinos/parasitología , Fasciola hepatica/genética , Fascioliasis/veterinaria , Variación Genética , Enfermedades de las Ovejas/parasitología , Animales , Carboxiliasas/análisis , Bovinos , ADN Polimerasa III/análisis , Fascioliasis/parasitología , Proteínas Fúngicas/análisis , Perú , Filogenia , Análisis de Secuencia de ADN , Ovinos , EspañaRESUMEN
Se efectuó un estudio con el empleo de la metodología de Gupta, en el Departamento de Biología y Geografía de la Universidad de Oriente en Santiago de Cuba, para determinar marcadores moleculares de tipo inserción en secuencias de las proteínas ADN polimerasa I y ADN polimerasa III (subunidad alfa), obtenidas de bases de datos internacionales y posteriormente alineadas con el programa ClustalX2. Las familias Moraxellaceae y Helicobacteraceae han sido ampliamente estudiadas, porque comprenden agentes patógenos causantes de numerosas enfermedades en humanos, pero pocas investigaciones han estado dirigidas a la identificación de las características moleculares que puedan distinguir a sus miembros de otros grupos de bacterias; de manera que los presentes resultados constituyen un aporte al conocimiento de la genética y la bioquímica de estas familias y proveen herramientas moleculares para la clasificación taxonómica y el diagnóstico de especies patógenas
A study with the use of Gupta methodology was carried out in the Biology and Geography Department of Oriente University in Santiago de Cuba, to determine molecular markers of insertion type in sequences of the DNA polimerase I proteins and DNA polimerase III (alpha subunity), obtained from international databases and later on aligned with the ClustalX2 program. The Moraxellaceae and Helicobacteraceae families have been broadly studied, because they comprise pathogen agents that cause numerous diseases in humans, but few investigations have been directed to the identification of the molecular characteristics that can distinguish their members from other groups of bacterias; so these results constitute a contribution to the knowledge of genetics and biochemistry of these families and provide molecular tools for the taxonomic classification and the diagnosis of pathogen species
Asunto(s)
Humanos , Helicobacter/citología , Moraxellaceae/citología , ADN Polimerasa I , ADN Polimerasa III , Biomarcadores , Marcadores Genéticos , Estudios EpidemiológicosRESUMEN
Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the ß clamp processivity factor by competing for binding to the ring. Moreover, the MutS-ß clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity.
Asunto(s)
ADN Polimerasa beta/metabolismo , Replicación del ADN , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Pseudomonas aeruginosa/metabolismo , Biocatálisis , ADN/biosíntesis , ADN/química , ADN Polimerasa III/metabolismo , Etilnitrosourea , Mutagénesis/genética , Péptidos/metabolismo , Unión Proteica , Pseudomonas aeruginosa/crecimiento & desarrollo , Respuesta SOS en Genética/genética , Especificidad por SustratoRESUMEN
The filamentous fungus Moniliophthora perniciosa is a basidiomycota that causes the witches' broom disease in cocoa trees (Theobroma cacao L.). The mitochondrial DNA polymerase of M. perniciosa (MpmitDNApol) is classified within the B family of DNA polymerases, which can be found in viruses and cellular organelles. Using virtual screening processes, accessing KEGG, PubChem, and ZINC databases, we selected the 27 best putative nucleoside viral-like polymerase inhibitors to test against MpmitDNApol. We used Autodock Vina to perform docking simulations of the selected molecules and to return energy values in several ligand conformations. Then, we used Pymol v1.7.4.4 to check the stereochemistry of chiral carbons, hydrogen bonding receptors, absence or presence of hydrogen, sub and superstructure, numbers of rings, rotatable bonds, and donor groups. We selected the Entecavir Hydrate, a drug used to control hepatitis B; subsequently AMBER 14 was used to describe the behavior of polymerase-entecavir complex after setting up 3500 ps of simulation in water at a temperature of 300 K. From the simulation, a graph of Potential Energy was generated revealing that the ligand remains in the catalytic site after 3500 ps with a final energy of -612,587.4214 kcal/mol.
Asunto(s)
Basidiomycota/genética , ADN Polimerasa III/genética , Hongos/genética , Enfermedades de las Plantas/genética , Biología Computacional , ADN Polimerasa III/aislamiento & purificación , Mitocondrias/genética , Enfermedades de las Plantas/virología , Interfaz Usuario-ComputadorRESUMEN
DNA replication is a key process in living organisms. DNA polymerase α (Polα) initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3) locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT) expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.
Asunto(s)
Arabidopsis/genética , ADN Polimerasa III/genética , Replicación del ADN/genética , Epigénesis Genética , Flores/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/biosíntesis , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Histonas/genética , Proteínas de Homeodominio/biosíntesis , Proteínas de Dominio MADS , Hojas de la Planta/genética , Factores de Transcripción/biosíntesisRESUMEN
DNA polymerase d is not only the major replicative enzyme in eukaryotic chromosomal DNA synthesis but is also the primary polymerase for most DNA repair pathways. However, the subunit composition of polymerase d varies in different organisms. While polymerase d in many eukaryotic species has all 4 subunits (POLD1, 2, 3, and 4), many other organisms do not possess POLD4. Whether POLD4 is indispensable and why these differences exist are unknown. In the present study, we identified the POLD4 protein sequences of 218 eukaryotic species and determined the POLD1, 2, and 3 protein sequences of 55 species representing various taxonomic groups. No insect and nematode species examined possessed POLD4. Approximately 80% of protozoan species did not contain POLD4. Nearly 50% of fungal species did not contain POLD4. Other animal and plant species are expected to contain POLD4. Phylogenetic analyses of POLD1, 2, 3, and 4 sequences revealed that most animal and plant species inherited DNA polymerase d from protozoa, whereas some other animal and plant species may have inherited polymerase d directly from fungi. Because a large number of protozoan and fungal species do not possess POLD4, current insect and nematode species lacking POLD4 may have evolved from ancestor protozoan species lacking POLD4; thus, other protozoan and animal species lacking POLD4 may share a similar evolutionary history. Future studies should examine the origin and indispensability of POLD4 in various organisms.
Asunto(s)
ADN Polimerasa III/genética , Eucariontes/enzimología , Eucariontes/genética , Genoma , Filogenia , Subunidades de Proteína/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada/genética , ADN Polimerasa III/química , Humanos , Datos de Secuencia Molecular , Subunidades de Proteína/química , Alineación de SecuenciaRESUMEN
Interaction between MutS and the replication factor ß clamp has been extensively studied in a Mismatch Repair context; however, its functional consequences are not well understood. We have analyzed the role of the MutS-ß clamp interaction in Pseudomonas aeruginosa by characterizing a ß clamp binding motif mutant, denominated MutSß, which does not interact with the replication factor. A detailed characterization of P. aeruginosa strain PAO1 harboring a chromosomal mutSß allele demonstrated that this mutant strain exhibited mutation rates to rifampicin and ciprofloxacin resistance comparable to that of the parental strain. mutSß PAO1 was as proficient as the parental strain for DNA repair under highly mutagenic conditions imposed by the adenine base analog 2-aminopurine. In addition, using a tetracycline resistance reversion assay to assess the repair of a frameshift mutation, we determined that the parental and mutSß strains exhibited similar reversion rates. Our results clearly indicate that the MutS-ß clamp interaction does not have a central role in the methylation-independent Mismatch Repair of P. aeruginosa.
Asunto(s)
ADN Polimerasa III/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Pseudomonas aeruginosa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , ADN Polimerasa III/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/química , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Mutación , Tasa de Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/genéticaRESUMEN
Increasing efforts are directed toward finding applications for natural products and their derivatives in the treatment of human diseases. Among such products, propolis, a resinous substance produced by honey bees from various plant sources, has been found to be a promising source of potential therapeutics. In the present work, we aimed at studying the perspective of Cuban propolis as a source of possible anti-cancer agents. We found an anti-metastatic effect in mice and considerable cytotoxicity without cross-resistance in both wild-type and chemoresistant human tumor cell lines. Plukenetione A--identified for the first time in Cuban propolis--induced G0/G1 arrest and DNA fragmentation in colon carcinoma cells. Furthermore, the activities of both topoisomerase I and DNA polymerase were inhibited, while the expression of topoisomerase II-beta, EGF receptor, and multidrug resistance-related protein genes was found repressed. We assume that plukenetione A contributes to the anti-tumoral effect of Cuban propolis mainly by targeting topoisomerase I as well as DNA polymerase.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Policíclicos/farmacología , Própolis/química , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Cuba , Daño del ADN/efectos de los fármacos , ADN Polimerasa III/antagonistas & inhibidores , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Fase G1/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Ratones , Compuestos Policíclicos/química , Compuestos Policíclicos/aislamiento & purificación , Própolis/farmacología , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Inhibidores de Topoisomerasa I , Células Tumorales CultivadasRESUMEN
DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.
Asunto(s)
Caulobacter crescentus/genética , Daño del ADN , Mutagénesis , Operón , Respuesta SOS en Genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Caulobacter crescentus/metabolismo , Caulobacter crescentus/efectos de la radiación , ADN Polimerasa III/clasificación , ADN Polimerasa III/genética , ADN Polimerasa III/fisiología , Genoma Bacteriano , Filogenia , Rayos UltravioletaRESUMEN
Contradictory biogeographic hypotheses for either a Neotropical or a Palaearctic origin of the genus Leishmania have been proposed. Hypotheses constructed on the basis of biogeographic data must be tested against an independent dataset and cannot be supported by biogeographic data alone. In the absence of a fossil record for the Leishmania these two hypotheses were tested against a combined dataset of sequences from the DNA polymerase A catalytic subunit and the RNA polymerase II largest subunit. The phylogeny obtained provided considerable support for a Neotropical origin of the genus Leishmania and leads us to reject the hypothesis for a Palaearctic origin.
Asunto(s)
Leishmania/genética , Filogenia , Animales , ADN Polimerasa III/análisis , Leishmania/clasificación , ARN Polimerasa II/análisisRESUMEN
The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.
Asunto(s)
Proteínas Bacterianas , Replicación del ADN/genética , Elementos Transponibles de ADN/genética , Escherichia coli/genética , Mutación , ADN Helicasas/genética , ADN Polimerasa III/genética , AdnB Helicasas , Escherichia coli/metabolismo , Genes Bacterianos , Genotipo , Modelos Genéticos , TemperaturaRESUMEN
Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.