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

Sivaraman Subramaniam,Michael Sattler,A Francis Stewart,Axel Erler,Daniel Müller,Adrian Keller,Andrea Kranz,Jing Tang,Jun Fu,Saminathan Ramakrishnan,Guido Grundmeier,Mohanraj Gopalswamy

doi:10.1038/srep34525

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.

Highlights

They share a similar protein architecture based on an N-terminal ssDNA binding domain of ~180 amino acids and a C-terminal extension that in the case of RAD52 is required for homologous recombination (HR) through specific protein-protein interactions[6]
RAD52 is the best characterized single strand annealing proteins (SSAPs) due, in part, to two crystal structures of the N-terminal DNA binding domain of human RAD5216,17. Both crystal structures revealed that the N-terminal domain of ~200 amino acids forms a mushroom-shaped undecameric ring with an external groove lined with positive charges, which probably binds the phosphodiester backbone of ssDNA
All Redβtruncations disabled recombination in both assays (Fig. 1d,f; data not shown) except for the least C-terminally truncated construct C1Redβ(1–237), which retained approximately 25% of the wt level in the single strand oligonucleotide repair (ssOR) assay as well as the expected bias between lagging and leading strands. These results indicate that the recombination functions of Redβrely on amino acids that lie outside of the conserved region at both ends of the protein and the very C-terminus is required for dsDNA but not ssDNA recombination

Summary

Introduction

In the absence of DNA in vitro, they multimerize into rings or chains at high concentrations (>0.5 μM) They share a similar protein architecture based on an N-terminal ssDNA binding domain of ~180 amino acids and a C-terminal extension that in the case of RAD52 is required for homologous recombination (HR) through specific protein-protein interactions[6]. These shared biochemical, protein sequence and functional similarities suggest the existence of an ancestral annealing mechanism involved in HR.

Methods

Results

Conclusion