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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.

Structural and functional characterization of the Redβ recombinase from bacteriophage λ Thumbnail


Authors

K. Matsubara

A.D. Malay

F.A. Curtis

J.G. Heddle



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
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.

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Publisher Licence URL
http://creativecommons.org/licenses/by/4.0/

Copyright Statement
© 2013 Matsubara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.






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