DNA translocation and regulation of single DNA helicase molecules

Investigation into the enzymatic mechanisms of various complex molecular motors such as kinesin, myosin, ncd and RNA polymerase has been facilitated by the advent of single-molecule techniques. Unlike conventional, macroscopic studies that reveal the population averaged properties of molecular ensembles, these methods monitor the stochastic properties of single enzyme molecules. To directly detect the movement of single Escherichia coli RNA polymerase molecules along a DNA template during active transcription, Schafer et al. (Nature 352: 444–448 (1991)) devised the tethered particle motion (TPM) method. In this thesis, I report the development of single-molecule TPM assays to study the Rep helicase and RecBCD helicase/nuclease of E. coli.; The poor processivity of the Rep helicase and an inability to ensure single enzyme molecule conditions limited the development of the Rep helicase TPM assay. Nonetheless, the assay developed provides the foundation for future single-molecule Rep helicase studies that should incorporate accessory proteins to increase the processivity of the enzyme. Conversely, the TPM assay developed using the RecBCD enzyme provides the first observation of single DNA helicase molecules translocating on double-stranded DNA in real time. RecBCD molecules initiate translocation upon binding a dsDNA end. Translocation is unidirectional, continuous and proceeds at a constant rate over the length of the DNA. The translocating enzyme molecules are kinetically homogeneous and move at the same rate as DNA unwinding, indicating that translocation and DNA unwinding are tightly coupled processes.; An ongoing controversy within the RecBCD field was whether recognition of the DNA sequence λ (5′-GCTGGTGG-3′ ) by the enzyme requires dissociation of the RecD subunit. Taking advantage of the experimental geometry of the developed TPM assay, which specifically tags the RecD subunit with a microscopic bead, we demonstrate that χ recognition does not require RecD release.; Finally, optical trapping techniques were used to determine the mechanical properties of RecBCD during translocation. Initial studies applying calibrated forces on the translocating enzyme show that RecBCD can withstand up to 9 pN of force. In addition, preliminary results suggest the presence of translocation steps that are 15–30 by in size.