Trans-complementation by the RecB nuclease domain of RecBCD enzyme reveals new insight into RecA loading upon χ recognition

Abstract

The loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a {chi}-dependent manner via its RecB nuclease domain (RecBn). Before recognition of {chi}, RecBn is sequestered through interactions with RecBCD. It was proposed that upon {chi}-recognition, RecBn undocks, allowing RecBn to swing out via a contiguous 70 amino acid linker to reveal the RecA-loading surface, and then recruit and load RecA onto ssDNA. We tested this hypothesis by examining the interactions between RecBn (RecB928-1180) and truncated RecBCD (RecB1-927CD) lacking the nuclease domain. The reconstituted complex of RecB1-927CD and RecBn is functional in vitro and in vivo. Our results indicate that despite being covalently severed from RecB1-927CD, RecBn can still load RecA onto ssDNA, establishing that RecBn does not function at the end of its flexible linker. Instead, RecBCD undergoes a {chi}-induced intramolecular rearrangement to reveal a RecA-loading surface.