RESUMO
Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1(-/-) mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1(-/-)/Csb(-/-)) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.
Assuntos
DNA Glicosilases/deficiência , Enzimas Reparadoras do DNA/deficiência , Reparo do DNA/fisiologia , DNA Mitocondrial/metabolismo , Guanosina/análogos & derivados , Animais , Corticoviridae/metabolismo , Dano ao DNA , DNA Glicosilases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Mitocondrial/química , DNA Viral/química , DNA Viral/metabolismo , Feminino , Guanosina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Extratos Vegetais/metabolismo , Proteínas de Ligação a Poli-ADP-RiboseRESUMO
Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.
Assuntos
Capsídeo/química , Membrana Celular/metabolismo , Corticoviridae/metabolismo , Vírion/química , Bacteriófagos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA Circular/química , Genoma , Genoma Viral , Processamento de Imagem Assistida por Computador , Metabolismo dos Lipídeos , Lipídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Pseudoalteromonas/virologiaRESUMO
Bacteriophage PM2 is the only described member of the Corticoviridae family. It is an icosahedral dsDNA virus with a membrane residing underneath the protein coat. PM2 infects some gram-negative Pseudoalteromonas spp. In the present study, we mapped the viral promoters and showed that the PM2 genome consists of three operons. Four new virus genes were assigned based on their function in transcription. Proteins P15 and P16 are shown to repress early transcription, and proteins P13 and P14 are shown to activate late transcription events. The early regulatory region, containing genes for proteins P15 and P16, as well as the newly identified early promoter region in PM2, has significant sequence similarity with the Pseudoalteromonas pAS28 plasmid. P14, the transcription activator for the structural genes, has a zinc finger motif homologous to archaeal and eukaryotic TFIIS-type regulatory factors.
Assuntos
Corticoviridae/genética , Regulação Viral da Expressão Gênica , Pseudoalteromonas/virologia , Transativadores/genética , Transcrição Gênica , Fatores de Elongação da Transcrição , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Corticoviridae/metabolismo , Primers do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Regiões Promotoras Genéticas , Pseudoalteromonas/genética , Transativadores/metabolismo , Fatores Genéricos de Transcrição/genética , Proteínas Virais/metabolismoRESUMO
Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.
