title: Deciphering the function of lactococcal phage ul36 Sak domains.
abstract: Virulent phages are responsible for milk fermentation failures in the dairy industry, due to their ability to infect starter cultures containing strains of Lactococcus lactis. Single-strand annealing proteins (SSAPs) have been found in several lactococcal phages, among which Sak in the phage ul36. Sak has been recently shown to be a functional homolog of the human protein RAD52, involved in homologous recombination. A comparison between full-length Sak and its N- and C-terminal domains was carried out to elucidate functional characteristics of each domain. We performed HPLC-SEC, AFM and SPR experiments to evaluate oligomerization states and compare the affinities to DNA. We have shown that the N-terminal domain (1-171) is essential and sufficient for oligomerization and binding to DNA, while the C-terminal domain (172-252) does not bind DNA nor oligomerize. Modelisation of Sak N-terminal domain suggests that DNA may bind a positively charged crevice that runs external to the ring. Annealing and stimulation of RecA strand exchange indicate that only the N-terminal domain is capable of single-strand annealing and both domains do not stimulate the RecA strand exchange reaction. We propose that Sak N-terminus is involved in DNA binding and annealing while the C-terminus may serve to contact Sak partners.
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: Sylvain
authors.initials: S
authors.lastname: Moineau
authors.forename: Hélène
authors.initials: H
authors.lastname: Launay
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Masson
authors.forename: Claudio
authors.initials: C
authors.lastname: Rivetti
authors.forename: Roberto
authors.initials: R
authors.lastname: Ramoni
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
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology; D001435:Bacteriophages / Q000737:chemistry* / Q000235:genetics / Q000472:pathogenicity; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D013294:Lactococcus lactis / Q000502:physiology / Q000821:virology*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D020349:Surface Plasmon Resonance; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
chemical_list: C106520:AbiK protein, Lactococcus lactis; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; C499652:RDM1 protein, human; D011994:Recombinant Proteins; D014764:Viral Proteins; D011938:Rec A Recombinases