New Insights into Redβ-mediated DNA Annealing using Atomic Force Microscopy

Redβ anneals DNA to initiate homologous recombination ined recent prominence through the development of the DNA engineering technology known as ‘recombineering' or ‘Red/ET'. It originates from the red-operon of λ phage where it is co-expressed in the early life cycle stage with Redα a processive 5'-3' exonuclease and Redγ, a DNA mimetic and RecBCD inhibitor. Unlike RecA/RAD51, Redβ is not an ATPase and it's mechanism for initiating homologous recombination is poorly understood. To examine the structure and dynamics of Redβ complexes at sub-molecular resolution we performed tapping mode atomic force microscopy (AFM) of Redβ protein alone and in complex with DNA. Without DNA, Redβ forms a ‘split lock washer' structure with a shallow right-handed helicity. Sequentially adding complementary ssDNA generates a stable left-handed helical filament. Importantly, the contour length of the helical filament equated linearly to the lengths of complementary ssDNA, giving the number of nucleotides per Redβ monomer. Additionally, the monomer width along the filament was quantified. These new quantities as well as the observed helical transition reveals new insights into the mechanism of DNA annealing mediated by Red and led us to suggest new mechanistic models.