title: Lactococcal phage p2 ORF35-Sak3 is an ATPase involved in DNA recombination and AbiK mechanism.
abstract: Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS and Universités d'Aix-Marseille I & II, Campus de Luminy, case 932, 13288 Marseille cedex 09, France.
authors.forename: Erika
authors.initials: E
authors.lastname: Scaltriti
authors.forename: Hélène
authors.initials: H
authors.lastname: Launay
authors.forename: Marie-Michelle
authors.initials: MM
authors.lastname: Genois
authors.forename: Patrick
authors.initials: P
authors.lastname: Bron
authors.forename: Claudio
authors.initials: C
authors.lastname: Rivetti
authors.forename: Stefano
authors.initials: S
authors.lastname: Grolli
authors.forename: Mickael
authors.initials: M
authors.lastname: Ploquin
authors.forename: Valérie
authors.initials: V
authors.lastname: Campanacci
authors.forename: Mariella
authors.initials: M
authors.lastname: Tegoni
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Sylvain
authors.initials: S
authors.lastname: Moineau
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Masson
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D017901:Bacteriophage P2 / Q000201:enzymology* / Q000235:genetics*; D018625:Microscopy, Atomic Force; D016366:Open Reading Frames / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C106520:AbiK protein, Lactococcus lactis; D001426:Bacterial Proteins; D014764:Viral Proteins; D000251:Adenosine Triphosphatases