Structure of a Rad52 homolog from bacteriophage in complex with a novel duplex intermediate of DNA annealing

Human Rad52 protein binds to ssDNA and promotes the annealing of complementary strands. This activity is central to multiple DNA repair pathways and Rad52 is a target for cancer therapeutics. Previous crystal structures of the DNA binding domain of Rad52 revealed an 11-mer ring that binds to ssDNA in an extended conformation with the bases exposed for homology recognition. While this complex is likely involved in the early stages of annealing, there is no structure of Rad52 with two strands of DNA bound simultaneously, and its mechanism of annealing is poorly understood. To approach this problem, we have turned to the RecT/Redβ family of annealing proteins from bacteriophage, which are distant homologs of Rad52 that form stable complexes with a duplex intermediate of annealing. We have used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a RecT homolog from a prophage of Listeria innocua (LiRecT) in complex with two complementary 83-mer oligonucleotides that were added to the protein sequentially. The structure reveals a left-handed helical filament of the protein bound to a novel conformation of DNA duplex that is highly extended and under-wound. The duplex is bound at a stoichiometry of 5 bp/monomer to a deep, narrow, positively-charged groove that runs along the outer surface of the filament. Data from native mass spectrometry confirm that the filament complex seen by cryo-EM also exists in solution. Collectively, these data provide new insights into the mechanism of annealing by LiRecT and by homologous proteins including human Rad52.