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Structural and functional characterization of the Red? recombinase from bacteriophage ?.

ABSTRACT: The Red system of bacteriophage ? is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Red?, a ring-shaped protein that facilitates annealing of complementary DNA strands. Red? shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Red? sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Red? residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Red? oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Red? can be separated. Taken together, the results provide fresh insights into the structural basis for Red? function and the important role of quaternary structure.

SUBMITTER: Matsubara K

PROVIDER: S-EPMC3823998 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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