title: Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.
abstract: The serine integrase of phage A118 catalyzes integrative recombination betweenattPon the phage and a specificattBlocus on the chromosome ofListeria monocytogenes, but it is unable to promote excisive recombination between the hybridattLandattRsites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the fourattsites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control whichattsites can synapse and recombine.IMPORTANCESerine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case ofListeria monocytogeneslysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Sridhar
authors.identifier: http://orcid.org/0000-0002-4970-0572
authors.initials: S
authors.lastname: Mandali
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Kushol
authors.initials: K
authors.lastname: Gupta
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Dawson
authors.affiliation: Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Van Duyne
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA rcjohnson@mednet.ucla.edu.|Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA.
authors.forename: Reid C
authors.initials: RC
authors.lastname: Johnson
mesh_terms: D020816:Amino Acid Motifs; D001435:Bacteriophages / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D015967:Gene Expression Regulation, Viral; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008087:Listeria / Q000821:virology*; D000072417:Protein Domains; D011995:Recombination, Genetic; D012694:Serine / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016662:Virus Integration
chemical_list: D014764:Viral Proteins; D012694:Serine; D019426:Integrases
keywords: Listeria monocytogenes; recombination directionality factor; serine integrase; site-specific DNA recombination