Structural and functional characterization of the Redβ recombinase from bacteriophage λ

Matsubara, K.; Malay, A.D.; Curtis, F.A.; Sharples, G.J.; Heddle, J.G.

Abstract

The Red system of bacteriophage λ 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 Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ 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 Redβ 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 Redβ 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 Redβ 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 Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.

Citation

Matsubara, K., Malay, A., Curtis, F., Sharples, G., & Heddle, J. (2013). Structural and functional characterization of the Redβ recombinase from bacteriophage λ. PLoS ONE, 8(11), Article e78869. https://doi.org/10.1371/journal.pone.0078869

• Journal Article Type: Article
• Acceptance Date: Sep 23, 2013
• Online Publication Date: Nov 11, 2013
• Publication Date: Nov 11, 2013
• Deposit Date: Sep 26, 2013
• Publicly Available Date: Jul 28, 2014
• Journal: PLoS ONE
• Electronic ISSN: 1932-6203
• Publisher: Public Library of Science
• Peer Reviewed: Peer Reviewed
• Volume: 8
• Issue: 11
• Article Number: e78869
• DOI: https://doi.org/10.1371/journal.pone.0078869
• Keywords: Bacteriophages, DNA annealing, DNA recombination, DNA-binding proteins, Homologous recombination, Recombinant proteins, Sequence alignment, Transmission electron microscopy.
• Public URL: https://durham-repository.worktribe.com/output/1477219