Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping essential for break repair. Yet, the energetics of RecBCD during translocation along the DNA track needs to be quantitatively clarified. Specifically, its essential to understand how RecBCD switches between its binding states to enable its translocation. Here we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. We show that, in the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to "sense" the nucleotide state. Excitingly, we observed weak coupling for AMPpNp as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of RecBCD reaction mechanism suggests an energetic compensation between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a stepping mechanism during the processive translocation of RecBCD.