Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

• Published in: Nucleic acids research Vol. 46; no. 3; pp. 1308 - 1320
• Main Authors: Fogg, Paul C M, Younger, Ellen, Fernando, Booshini D, Khaleel, Thanafez, Stark, W Marshall, Smith, Margaret C M
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 16.02.2018
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Attachment Sites, Microbiological; Binding Sites; Cloning, Molecular; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Integrases - chemistry; Integrases - genetics; Integrases - metabolism; Lysogeny; Models, Molecular; Molecular Biology; Mutation; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Siphoviridae - chemistry; Siphoviridae - genetics; Siphoviridae - metabolism; Streptomyces - chemistry; Streptomyces - virology; Thermodynamics; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx1233

Abstract 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.