DNA annealing by Red? is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions.

ABSTRACT: Single strand annealing proteins (SSAPs) like Red? initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Red?, 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 Red?-Red? 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 Red? 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 Red?-host factor interaction. These data further advance the model for Red recombination and the proposition that Red? and RAD52 SSAPs share ancestral and mechanistic roots.

SUBMITTER: Subramaniam S 

PROVIDER: S-EPMC5052646 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature