Abstract
In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system.
Highlights
The repair of double-strand DNA breaks is a critical process in all organisms
One of the major pathways for high fidelity repair of breaks is homologous recombination initiated by RecBCD, AdnAB or AddAB complexes (Dillingham and Kowalczykowski, 2008; Wigley, 2013) that process the broken ends via a number of steps to eventually produce a RecA-coated 3’-tailed filament that initiates strand invasion
In addition to a role in repair of double-strand DNA breaks, RecBCD is an important player in controlling infection by bacteriophages, the significance of which is exemplified by proteins produced by phages that target RecBCD
Summary
The repair of double-strand DNA breaks is a critical process in all organisms. In bacteria, one of the major pathways for high fidelity repair of breaks is homologous recombination initiated by RecBCD, AdnAB or AddAB complexes (Dillingham and Kowalczykowski, 2008; Wigley, 2013) that process the broken ends via a number of steps to eventually produce a RecA-coated 3’-tailed filament that initiates strand invasion. There has been considerable study of RecBCD at the biochemical and single molecule level (Wright et al, 1971; Taylor and Smith, 1992, 2003; Dillingham and Kowalczykowski, 2008; Spies et al, 2003, 2007), our understanding of the molecular details for the RecBCD mechanism are less advanced due to a lack of high resolution structural information To date, this has been limited to the initiation complex, with the protein interacting with a broken DNA end A critical role for a rare bacterial SH3 domain in the RecD subunit is revealed in the structure, and raises possibilities for a mechanism of further regulation after recognition of Chi
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