Abstract
Double strand breaks are the severest genomic damage requiring rapid repair response. In prokaryotes, members of the RecBCD family initiate DNA unwinding essential for double strand break repair mechanism by homologous recombination. RecBCD is a highly processive DNA helicase with an unwinding rate approaching ∼1,600 bp·s-1. The ATPase reaction mechanism enabling RecBCD to achieve this fast unwinding rate and its enzymatic adaptation are not fully understood. Here, we present thermodynamic investigation of DNA and nucleotide binding to RecBCD to reveal the binding linkage and the degree of coupling between its nucleotide cofactor and DNA substrate binding. We find that RecBCD exhibits a weak binding state in the presence of ADP towards double overhang DNA substrate (dohDNA), and the same degree of coupling is observed for RecBCD affinity toward ADP, only in the presence of dohDNA. In the absence of nucleotide cofactor (APO state) or in the presence of AMPpNp, much weaker coupling is observed between the binding of DNA and the nucleotide state towards RecBCD. Other DNA substrates that are not optimally engaged with RecBCD do not exhibit similar degree of coupling. This may be the first evidence for strong and weak binding states that can, in principle, regulate a ‘stepping mechanism’ during processive translocation of RecBCD.
