title: DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions.
abstract: Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots.
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Sivaraman
authors.initials: S
authors.lastname: Subramaniam
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Erler
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.|Shandong University-Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Kranz
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: Jing
authors.initials: J
authors.lastname: Tang
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany and Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747 Garching, Germany.
authors.forename: Mohanraj
authors.initials: M
authors.lastname: Gopalswamy
authors.affiliation: Technical and Macromolecular Chemistry, University of Paderborn, Warburger Str. 100 33098 Paderborn, Germany.
authors.forename: Saminathan
authors.initials: S
authors.lastname: Ramakrishnan
authors.affiliation: Technical and Macromolecular Chemistry, University of Paderborn, Warburger Str. 100 33098 Paderborn, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Keller
authors.affiliation: Technical and Macromolecular Chemistry, University of Paderborn, Warburger Str. 100 33098 Paderborn, Germany.
authors.forename: Guido
authors.initials: G
authors.lastname: Grundmeier
authors.affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich, Mattenstraße 26, 4058 Basel, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Müller
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany and Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technische Universität München, Lichtenbergstr.4, 85747 Garching, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Sattler
authors.affiliation: Genomics, Biotechnology Center, TU Dresden, Tatzberg 47/49, 01307 Dresden, Germany.
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination
chemical_list: D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins