Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

The bacteriophage lambda red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Red-Beta annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Red-Beta with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 Angstrom. This structure validates and rationalizes decades of experimental observations on the biochemistry of Red-Beta. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Red-Beta, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. Residues 133-138, which are missing from our structure, form a flexible loop. Molecular dynamics simulations were used to model the range of motion of the flexible loop, which suggested that it has a crucial role in keeping the DNA strands in the DNA-binding groove long enough to allow homology searching. The combination of structural and computational observations has allowed us to propose a detailed mechanism for the action of Red-Beta. More than half a century after its discovery, our work shines a light not only on the structure and mechanisms of Red-Beta, but also of other proteins within the annealase superfamilies. ### Competing Interest Statement The authors have declared no competing interest.