Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

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.

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

“…We speculate that the protein products of orf8 and orf9 may form a complex, like other two-component recombinase pairs, such as Exo/beta protein from coliphage (Red system), RecE/T from the Rac prophage of E. coli, YqaJ/K from the skin prophage of Bacillus subtilis, and G34.1P/G35P from the phage SPP1. Such complexes contribute to the activity of homologous recombination with linear dsDNA and are independent of the host recombination proteins (43). Homologous recombination has been proposed to be one of the pathways for generating substrates with the correct topology (genomic concatemers) for packaging into infectious viral particles (44).…”

Bacteriophage lambda: Early pioneer and still relevant

“…These mutations were found to affect genes exo and bet that lie in the early P L transcript. Exo is an exonuclease (Zhang et al , 2011 and refs therein) and Bet is a strand-annealing protein (Matsubara et al , 2013 and refs therein). These two proteins can substitute for RecA in homologous recombination.…”

Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

“…Although β (29 kDa) and RecT (33 kDa) share little overall sequence identity, they contain conserved residues that are also present in Rad52, a SSAP that functions in homologous recombination in eukaryotes (32, 83). Electron microscopy (EM) and atomic force microscopy (AFM) studies have suggested that β forms rings of ~12 subunits in the absence of DNA (96).…”