Architecture of recombination intermediates visualized by in-gel FRET of λ integrase–Holliday junction–arm DNA complexes

摘要

λ Integrase (Int) mediates recombination between attachment sites on phage and Escherichia coli DNA. Int is assisted by accessory protein-induced DNA loops in bridging pairs of distinct “arm-type” and “core-type” DNA sites to form synapsed recombination complexes that subsequently recombine by means of a Holliday junction (HJ) intermediate. An in-gel FRET assay was developed and used to measure 15 distances between six points in two Int–HJ complexes containing arm-DNA oligonucleotides, and 3D maps of these complexes were derived by distance-geometry calculations. The maps reveal unexpected positions for the arm-type DNAs relative to core sites on the HJ and a new Int conformation in the HJ tetramer. The results show how the position of arm DNAs determines the bias of catalytic activities responsible for directional resolution, provide insights into the organization of Int higher-order complexes, and lead to models of the structure of the full HJ recombination intermediates.