Abstract
The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA-binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.
Highlights
The homologous recombination (HR) pathway, responsible for the repair of double-strand breaks (DSBs), is essential to preserve the integrity of the genome and exists in all domains of life (Sung and Klein, 2006)
In Escherichia coli, the HR process is initiated by RecBCD, which binds to the damage site and unwinds the DNA, in preparation for strand invasion (Dillingham and Kowalczykowski, 2008)
This assay is not appropriate for the study of RecBCD since, in line with its biological function that requires binding to DSB sites, RecBCD’s affinity towards DNA lacking a double stranded end is 106-fold weaker than that for blunt ends or short overhangs (Bianco et al, 2001; Roman and Kowalczykowski, 1989a; Taylor and Smith, 1985)
Summary
The homologous recombination (HR) pathway, responsible for the repair of double-strand breaks (DSBs), is essential to preserve the integrity of the genome and exists in all domains of life (Sung and Klein, 2006). In Escherichia coli, the HR process is initiated by RecBCD, which binds to the damage site and unwinds the DNA, in preparation for strand invasion (Dillingham and Kowalczykowski, 2008). The RecC subunit is a 128 kDa, catalytically dead helicase-nuclease sharing a similar structure to RecB, which acts as a scaffold protein stapling RecB and RecD (Dillingham and Kowalczykowski, 2008), and is responsible for recognition of the regulatory Chi sequence (Handa et al, 2012; Singleton et al, 2004). The RecA nucleofilament is used to invade the acceptor DNA
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