1. Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model.
- Author
-
Amundsen SK and Smith GR
- Subjects
- Adenosine Triphosphatases metabolism, DNA metabolism, Escherichia coli enzymology, Signal Transduction, DNA Helicases genetics, DNA Helicases metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Exodeoxyribonuclease V chemistry
- Abstract
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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF