bioRxiv Biochemistry
bioRxiv Biochemistry
5 years ago

RecBCD possesses strong coupling between DNA and nucleotides binding that may propel stepping mechanism during translocation

O., Dvir, V., R., Gaydar, Kaplan, Zananir, A., Henn
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{middle dot}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 linkage binding and the degree of coupling between its nucleotides cofactor and DNA substrates binding. We find that RecBCD exhibits 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 nucleotides state towards RecBCD. Other DNA substrates that do not fully engaged with RecBCD optimally, do not exhibits similar degree of coupling. This may be the first evidence for strong and weak binding states that can in principle regulate stepping mechanism during processive translocation of RecBCD.

Publisher URL: http://biorxiv.org/cgi/content/short/190215v1

DOI: 10.1101/190215

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