The goal of this research proposal is to provide insight into the biochemical mechanisms ofDNA helicases at the single molecule level. To achieve this goal, this proposal is subdivided into three primary questions. By what mechanism do structurally distinct helicases facilitate processing of complex DNA substrates? How do the various helicase motifs achieve translocation and strand separation? What are the forces exerted by DNA motors as they proceed to translocate and unwind DNA? For the majority of experiments, single molecule techniques will be used to answer these three questions. To provide answers to the first two questions, we will combine optical tweezers and fluorescence microscopy to directly visualize these dynamic nanomachines in motion. The experiments designed to address question three will take advantage of atomic force microscopy to measure the forces in operation for DNA helicases during translocation and DNA unwinding. We will use these single molecule techniques to study four DNA motor proteins that have been selected to provide both unique and complementary information on the details and nuances of the dynamics of motion of these enzymes. These Escherichia coli enzymes are the DNA helicases RecBCD, RecG, RuvAB and the type I restriction enzyme EcoR124I. It is anticipated that direct observation of these nanomachines in real time will provide novel insights into the biochemical mechanism of DNA helicases, a class of nucleic acid motors that are of fundamental importance to DNA metabolism. In addition, a more detailed understanding of these proteins will contribute to a general appreciation of the molecular events responsible for aberrant DNA metabolic processes. The importance of studying DNA helicases is emphasized by evidence demonstrating that the genetic defects leading to Bloom's syndrome, Cockayne's syndrome, Werner syndrome, and xeroderma pigmentosum, have all been identified as mutations in DNA helicases.
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