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

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.

Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

“…Expression from the T7 promoter was induced at mid-exponential growth phase with 0.2 mM IPTG at 20°C overnight. His 6 -tagged (56) and MBP-tagged (40) proteins were purified as described previously. All chromatography steps were carried out on an AKTA Prime instrument (GE Healthcare).…”

Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination

“…An RDF-independent mutant of ϕC31 integrase, Int(E449K), which can catalyze attL–attR recombination by itself [ 79 ], is available. Furthermore, a fusion protein between the integrase and RDF has been shown to follow the same regulation of recombination directionality as that established by RDF added in trans to the wild type integrase [ 80 ]. Analysis of the RDF-independent integrase and the hybrid integrase-RDF by TPM would be useful in providing additional insights into the specific steps of recombination controlled by RDF.…”

Extreme C‐terminal element of SprA serine integrase is a potential component of the “molecular toggle switch” which controls the recombination and its directionality

“…Accumulating evidence suggests that the CC motif of LSRs also participates in recombination directionality (Fogg et al, 2017; Gupta et al, 2017; Rowley et al, 2008; Rutherford & Van Duyne, 2014). The CC motif is composed of two antiparallel α‐helices projecting from the zinc‐ribbon domain.…”

“…The CC motif can form a homodimer by hydrophobic interaction between the α‐helices (CC–CC interaction) (McEwan et al, 2009). The CC–CC interaction between two recombination subunits ( attP ‐LSR dimers and attB ‐LSR dimers) is required for assembly of the subunits (Fogg et al, 2017; Mandali et al, 2017; Rutherford et al, 2013). The geometry of the CC motif provides structural complementarity between the attP ‐LSR and attB ‐LSR dimers, permitting synapsis of the subunits through inter‐subunit CC–CC interaction.…”