Monitoring the Individual Motor Activities of RecB and RecD within the E.coli Recbcd Helicase

Abstract

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO.E. coli RecBCD is a bipolar DNA helicase consisting of two superfamily-1 motor subunits: RecB (3’ to 5’ directionality) and RecD (5’ to 3’ directionality). Although these subunits have opposite translocation polarities, they function in unison and unwind duplex DNA in the same net direction by acting on opposite ends of the nucleic acid. The activity of RecBCD is controlled by an 8 nucleotide sequence within the unwound ssDNA, called chi. Before interacting with this chi site, the RecD motor moves faster than RecB. After chi, these relative rates are switched. However, it has been difficult to examine the coupling between the two motors by measuring DNA unwinding activity alone. Recently, we have developed a rapid kinetic assay (Wu et al (2010) Nature Structural & Molecular Biology (doi:10.1038/nsmb.1901) that allows us to monitor independently the translocation activity of the RecB motor and the RecD motor within a functioning RecBCD helicase. Using this approach we have examined the activities of these two motors to determine the extent to which they are coupled within an active RecBCD enzyme. (supported by NIH GM045948 to TML).