RESUMO
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.
Assuntos
Basidiomycota/genética , DNA Polimerase III/genética , Fungos/genética , Doenças das Plantas/genética , Biologia Computacional , DNA Polimerase III/isolamento & purificação , Mitocôndrias/genética , Doenças das Plantas/virologia , Interface Usuário-ComputadorRESUMO
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.
Assuntos
Arabidopsis/genética , DNA Polimerase III/genética , Replicação do DNA/genética , Epigênese Genética , Flores/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/biossíntese , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Histonas/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Domínio MADS , Folhas de Planta/genética , Fatores de Transcrição/biossínteseRESUMO
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.
Assuntos
DNA Polimerase III/genética , Eucariotos/enzimologia , Eucariotos/genética , Genoma , Filogenia , Subunidades Proteicas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada/genética , DNA Polimerase III/química , Humanos , Dados de Sequência Molecular , Subunidades Proteicas/química , Alinhamento de SequênciaRESUMO
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.
Assuntos
DNA Polimerase III/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pseudomonas aeruginosa/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , DNA Polimerase III/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Mutação , Taxa de Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/genéticaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Compostos Policíclicos/farmacologia , Própole/química , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cuba , Dano ao DNA/efeitos dos fármacos , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Feminino , Fase G1/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Camundongos , Compostos Policíclicos/química , Compostos Policíclicos/isolamento & purificação , Própole/farmacologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Inibidores da Topoisomerase I , Células Tumorais CultivadasRESUMO
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.
Assuntos
Caulobacter crescentus/genética , Dano ao DNA , Mutagênese , Óperon , Resposta SOS em Genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Caulobacter crescentus/metabolismo , Caulobacter crescentus/efeitos da radiação , DNA Polimerase III/classificação , DNA Polimerase III/genética , DNA Polimerase III/fisiologia , Genoma Bacteriano , Filogenia , Raios UltravioletaRESUMO
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.