When their DNA is broken, cells must repair it or they die. Faithful repair occurs by the reactions that can promote homologous recombination if the cell is heterozygous for genetic markers. The enzymatic reactions of homologous recombination are thus important for continuation of life of the individual and for evolution of the species. The major pathway of repair of DNA double-strand breaks (DSBs) and recombination in Escherichia coli requires the large, multifunctional RecBCD enzyme.1, 2, 3, 4 This complex, three-subunit enzyme has eight known enzymatic activities, including two ATP-dependent helicases of opposite DNA strand polarities: slower 3′ → 5′ translocation by RecB and faster 5′ → 3′ translocation by RecD.5, 6 RecB also contains a nuclease domain, and RecC contains a tunnel through which the 3′-ended strand passes and in which the Chi recombination hotspot (5′-GCTGGTGG-3′) is recognized during rapid DNA unwinding (Figure 1A).7, 8, 9, 10

After encountering Chi, RecBCD′s activities are dramatically changed in a manner that requires proper interaction and co-ordination among the three subunits. RecBCD cleaves the strand with Chi a few nucleotides to the 3′ side of Chi and begins to load the DNA strand-exchange protein RecA onto this newly generated 3′-ended single-stranded DNA (Figure 1B).11, 12, 13 DNA unwinding transiently stops for a few seconds and then continues at a slower rate.14 After nicking DNA at Chi, RecBCD loses the ability to cut at a subsequently encountered Chi, either on the same DNA or on another DNA molecule.15 Failure to cut on the same DNA can account for the reduction of intracellular Chi hotspot activity by another Chi site inserted in cis.16 At some point, such as at the end of the DNA substrate for purified RecBCD, the three subunits dissociate and remain inactive for an hour or more.17 This inactivation is Chi-dependent and can account for Chi′s action in trans in cells (i.e., on another DNA molecule).17, 18, 19 Conformational changes are also induced by Chi, as evidenced by RecBCD′s increased sensitivity, at limited sites, to proteases.20 These sites are protease-sensitive before DNA binding, become more resistant upon DNA binding and during initial unwinding, and become sensitive again after Chi′s encounter during continued unwinding.

These multiple responses to Chi require the RecC tunnel (for Chi recognition) and RecB (for Chi nicking). RecD is also required because recD nonsense and deletion mutants lack Chi′s stimulation of recombination (Chi hotspot activity), Chi nicking activity, and all detectable nuclease activity, but they retain DNA unwinding activity.21, 22 These and other observations led to the “intramolecular signal transduction” model (Figure 1C).23, 24 In this model, when Chi is recognized in the RecC tunnel, RecC signals RecD to stop unwinding. This cessation of unwinding, in turn, prompts RecD to signal the RecB nuclease domain (Nuc) to nick the DNA near Chi. This model is supported by mutations altering each of the points of intersubunit contact,24 determined in crystal and cryoEM structures,8, 9, 10, 25 between RecC-RecD, RecD-RecB, and RecB-RecC. These mutants lack Chi hotspot activity and presumably block the signal transduction at the indicated steps (Figure 1C). The related nuclease swing model (Figure 1D), which accounts for the changes of protease-sensitivity noted above, is supported by mutations in the 19-amino-acid tether connecting Nuc to the RecB helicase domain and in the 40-amino-acid “RecC loop” (Figure 1A) at which Nuc is postulated to be in its inactive state during unwinding before Chi.20, 26, 27 The nuclease swing model is also supported by physical analyses – the limited proteolysis noted above and small angle X-ray scattering (SAXS) analysis showing, upon RecBCD′s binding DNA, movement of mass from the RecC tunnel exit to the RecC loop (Figure 1D).20

Here, we test the signal transduction model further by employing enzymatic analyses. We report our discovery that the ATP analog ATPγS induces RecBCD to nick the DNA at a novel position, likely by the mechanism previously proposed for the small-molecule inhibitor NSAC1003 and two RecB ATPase-site mutants (see Results section).23, 28 RecBCD mutants altered in points of direct contact between subunit pairs,24 such as RecC-RecD for step 2 and RecD-RecB for step 3 (Figure 1C), differentially respond to these inhibitors and the RecB ATPase-site mutations, in the manner predicted by the signal transduction model. These enzymatic tests complement our previous genetic tests23, 24, 26 and further support this model for the control of a complex, multisubunit enzyme. Our approaches may be useful in elucidating control mechanisms in other complex enzymes with multiple activities and subunits.