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

Abstract

SummaryThe loading of RecA onto ssDNA by RecBCD is an essential step of RecBCD-mediated homologous recombination. RecBCD facilitates RecA-loading onto ssDNA in a χ-dependent manner via its RecB nuclease domain (RecBn). Before recognition of χ, RecBnis sequestered through interactions with RecBCD. It was proposed that upon χ-recognition, RecBnundocks, allowing RecBnto 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 RecBnis functionalin vitroandin vivo. Our results indicate that despite being covalently severed from RecB1–927CD, RecBncan still load RecA onto ssDNA, establishing that RecBndoes not function at the end of its flexible linker. Instead, RecBCD undergoes a χ-induced intramolecular rearrangement to reveal a RecA-loading surface.