Single-Molecule Assembly of Protein-DNA Complexes

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DESCRIPTION (provided by applicant): The major objective of this grant proposal is to develop a new experimental technique for the study of protein-DNA interactions at the single-molecule level. This proposal describes a novel instrument to study the dynamics and function of individual complexes of proteins and DNA. The design will permit the direct visualization by fluorescence microscopy, in real-time, of the assembly, disassembly, and movement of proteins on single, optically-trapped DNA molecules using a novel, multi-port, laminar-flow, micro machined flow-cell. This instrument will allow us to readily introduce an individual, optically-trapped DNA molecule (or protein-DNA complex) sequentially to other proteins and/or reaction conditions, and visualize the changes in structure/assembly of the molecules in real-time using multi-wavelength fluorescence microscopy. The instrument will also measure the forces generated by these protein-DNA interactions. Several different protein-DNA complexes will be examined; each is an essential component of genetic recombination. Two classes of proteins that will be examined are the DNA motor proteins known as helicases, and the DNA strand exchange proteins known as recombinases. The DNA helicases central to this process in Escherichia coli are the RecBCD enzyme and the RecQ helicase. The recombinases essential to this process are RecA (prokaryotes) and Rad51 (eukaryotes) proteins, and their ancillary protein components. This grant proposal has two specific aims. The first is to develop an instrument that will permit analysis of individual assemblies of protein-DNA complexes. The second broad objective is to examine the formation, dissociation, and translocation of the several specific DNA helicases (motor proteins) and DNA-enzyme complexes (protein machines) that are involved in genetic recombination and DNA repair. Development of this instrument will enable new types of single-molecule experiments, experiments that will uncover information about the dynamics and function of protein-DNA interactions that cannot be obtained from large ensembles of DNA molecules.