DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions

Abstract

Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots.

Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red‐beta annealase

“…23 Finally, through crosslinking Redβ to a 36mer oligonucleotide, DNA-binding was localized to a 20 kDa N-terminal domain (NTD). 21 In 2016, 10 years after identification of the NTD of Redβ as the DNA-binding domain, Smith and Bell 24 and Subramaniam et al 25 independently investigated the functional roles of the N and C-terminal domains (CTDs) in detail. Smith and Bell 24 examined the effect of DNA length on the binding affinity for WT and C-terminally truncated (residues 1-177) Redβ.…”

“…Attention was then turned to the CTD of Redβ, with circular dichroism spectroscopy experiments by Smith and Bell 24 predicting that residues 182-261 formed a predominantly α-helical fold, which was sufficient to form a stable interaction with λExo. Subramaniam et al 25 reported a K D of 8 μM for the interaction between full-length Redβ and λExo, but also found it was weakened by deletion to the NTD domain, suggesting that the NTD may still have some influence on λExo binding, and hence this point requires further clarification. Both groups then went on to show that while still able to affect annealing, C-terminally truncated Redβ was unable to mediate recombination 24,25 and that the removal of just 11 amino acids from the C-terminus 24 or the introduction of point mutations 25 prevented recombination.…”

“…Subramaniam et al 25 reported a K D of 8 μM for the interaction between full-length Redβ and λExo, but also found it was weakened by deletion to the NTD domain, suggesting that the NTD may still have some influence on λExo binding, and hence this point requires further clarification. Both groups then went on to show that while still able to affect annealing, C-terminally truncated Redβ was unable to mediate recombination 24,25 and that the removal of just 11 amino acids from the C-terminus 24 or the introduction of point mutations 25 prevented recombination. However, Smith and Bell 24 hypothesized that abolition of recombination was not due to disruption of the Redβ (via the CTD)-λExo interaction.…”

“…49 A modest K D of 10 μM was calculated, which was similar to that previously determined for Redβ binding to λExo (K D of 7.9 μM). 25 Though the similarity in the two binding affinities may be coincidental, binding interactions in the low micromolar range simultaneously allow for specific yet dynamic protein interactions. Moreover, further mutagenesis experiments suggested that the SSB and λExo binding sites may at least partially overlap.…”

“…This model would explain the loss of recombination in vivo (see above) that was observed with C-terminally mutated or truncated Redβ. 24,25…”