Mutational Analysis of Redβ Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo

• Main Authors: Katerina Zakharova, Brian J. Caldwell, Shalya Ta, Carter T. Wheat, Charles E. Bell
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: International Journal of Molecular Sciences
• Subjects: single-strand annealing; DNA recombination; DNA repair; homologous recombination; bacteriophage λ; recombineering
• Online Access: https://www.mdpi.com/1422-0067/22/14/7758
• ISSN: 1661-6596; 1422-0067

Redβ is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redβ, models of its DNA binding domain (residues 1–188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and <i>E. coli</i> single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redβ were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.