Assuntos
Bacteriófagos/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófagos/enzimologia , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/virologia , Dados de Sequência Molecular , Plasmídeos , Alinhamento de Sequência , Transativadores/genéticaRESUMO
The N15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends. Upon infection, the phage DNA circularizes via its cohesive ends, after which a specific enzyme, the N15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres. Protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecules. We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter. We also found the transformation efficiency of E. coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size. At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids. We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases.
Assuntos
Bacteriófagos/genética , Precursores Enzimáticos/fisiologia , Regulação Viral da Expressão Gênica , Telomerase/fisiologia , Proteínas Virais/fisiologia , Bacteriófagos/metabolismo , Sequência de Bases , Precursores Enzimáticos/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Telomerase/genética , Proteínas Virais/genética , Replicação ViralRESUMO
The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.
Assuntos
Colífagos/enzimologia , Colífagos/genética , DNA Viral/química , Precursores Enzimáticos/metabolismo , Conformação de Ácido Nucleico , Telomerase/metabolismo , Proteínas Virais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Colífagos/fisiologia , Replicação do DNA , DNA Viral/biossíntese , DNA Viral/genética , Dimerização , Precursores Enzimáticos/deficiência , Precursores Enzimáticos/genética , Escherichia coli/virologia , Genes Virais/genética , Modelos Genéticos , Plasmídeos/genética , Provírus/química , Provírus/genética , Provírus/crescimento & desenvolvimento , Replicon/genética , Telomerase/deficiência , Telomerase/genética , Replicação ViralRESUMO
Investigations carried out demonstrated a possibility of transmission of plasmid Rldrd19 from E. coli to Hafnia. The incidence of the plasmid transmission varied from 10(-7) to 10(-9) and depended on the properfies of Hafnia strains. Tra-operon of plasmid Rldrd19 in the Hafnia 614 strain functioned with the same efficacy as in E. coli. Plasmid Rldrd19 in Hafnia was unstable and was eliminated from the cells in case of storage at low temperatures. As shown, plasmid Rldrd19 was under strict replication control in Hafnia as in E. coli. Formation of the CCC-form of the plasmid Rldrd19 was suppressed in Hafnia at 29--30 degrees C and was not suppressed in E. coli.
