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1.
Nucleic Acids Res ; 46(3): 1308-1320, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29228292

RESUMO

To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase.


Assuntos
Sítios de Ligação Microbiológicos , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Integrases/química , Siphoviridae/genética , Streptomyces/virologia , Proteínas Virais/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Integrases/genética , Integrases/metabolismo , Lisogenia , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Siphoviridae/química , Siphoviridae/metabolismo , Streptomyces/química , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Mol Microbiol ; 80(6): 1450-63, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21564337

RESUMO

The serine integrase, Int, from the Streptomyces phage φC31 mediates the integration and excision of the phage genome into and out of the host chromosome. Integrases usually require a recombination directionality factor (RDF) or Xis to control integration and excision and, as φC31 Int only mediates integration in the absence of other phage proteins, we sought to identify a φC31 RDF. Here we report that the φC31 early protein, gp3 activated attL x attR recombination and inhibited attP x attB recombination. Gp3 binds to Int in solution and when Int is bound to the attachment sites. Kinetic analysis of the excision reaction suggested that gp3 modifies the interactions between Int and the substrates to form an active recombinase. In the presence of gp3, Int assembles an excision synaptic complex and the accumulation of the integration complex is inhibited. The structure of the excision synaptic complex, like that of the hyperactive mutant of Int, IntE449K, appeared to be biased towards one that favours the production of correctly joined products. The functional properties of φC31 gp3 resemble those of the evolutionarily unrelated RDF from phage Bxb1, suggesting that these two RDFs have arisen through convergent evolution.


Assuntos
Integrases/metabolismo , Recombinação Genética , Fagos de Streptococcus/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação Microbiológicos , Escherichia coli/virologia , Integrases/genética , Dados de Sequência Molecular , Ligação Proteica , Fagos de Streptococcus/enzimologia , Fagos de Streptococcus/genética , Proteínas Virais/genética , Integração Viral
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