Investigating the Role of the Auxiliary Nucleotide Binding Sites in the Recbcd DNA Helicase

Abstract

RecBCD is highly processive and very fast DNA helicase-nuclease, responsible for the initiation of double-stranded break repair in E. coli. By employing a combination of equilibrium and time-resolved unwinding and binding experiments, both ensemble and single-molecule, we previously demonstrated the existence of auxiliary binding sites in RecBCD, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. We showed that RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites located in RecB and RecD subunits. Equilibrium dialysis indicated the number of nucleotide-binding sites to be at least four, and their likely location in the RecC subunit. In this current work, we provide further support for the functional role of the auxiliary sites, by abolishing the nucleotide-binding sites in RecC. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking and mass spectrometry, as well as molecular docking studies. Based on these predictions, several mutations in RecC were carefully designed, producing unique RecBCmutD. We have successfully cloned and purified RecBCmutD and shown that RecBCmutD has DNA-activated ATPase activity. Furthermore, an equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same binding isotherm as the WT RecBCD. Based on these observations, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.