Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model

[Display omitted] •● Chi hotspots contol RecBCD enzyme via complex interactions among its three subunits.•● Small molecules and RecB ATPase-site mutations convert RecBCD into a Chi-activated state.•● Chi-activated state requires steps 3 and 4, but not 1 or 2, of the signal transduction model.•● Enzymatic results here and previous genetic results strongly support the model.•● The approaches used here provide a paradigm for studying other complex enzymes. Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5′-GCTGGTGG-3′). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model – upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.