TY - JOUR T1 - Structure of phage λ Redβ<sub>177</sub> annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination JF - bioRxiv DO - 10.1101/2022.04.09.487726 SP - 2022.04.09.487726 AU - Timothy Newing AU - Jodi L. Brewster AU - Haibo Yu AU - Nikolas P. Johnston AU - Lucy J. Fitschen AU - Gökhan Tolun Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/04/09/2022.04.09.487726.abstract N2 - The bacteriophage λ red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Redβ annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Redβ with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 Å. This structure validates and rationalizes decades of experimental observations on the biochemistry of Redβ. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Redβ, 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 Redβ. More than half a century after its discovery, our work shines a light not only on the structure and mechanisms of Redβ, but also of other proteins within the annealase superfamilies.Significance Statement Single-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Redβ protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease λExo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Redβ, providing details about the mechanism of homologous DNA annealing.Competing Interest StatementThe authors have declared no competing interest. ER -