Single-molecule approaches can be used to achieve a fundamental understanding of biological processes occurring at the nanometer (nm) scale, in the millisecond (ms) time domain and at the piconewton (pN) force level. In this study, we will develop novel uses of single-pair Fluorescence Resonance Energy Transfer (spFRET), Magnetic Tweezers (MT) and combination instruments for the analysis of DNA-structure specific helicases, essential motor proteins for life. We will develop combination scanning confocal fluorescence microscope/magnetic tweezers (SCFM/MT) to study interactions between helicase and helicase-protein complexes and DNA, and a scanning confocal fluorescence microscope (SCFM) that can follow spFRET of two dye pairs on the same molecule. We will also develop a horizontal MT to follow tether length changes of the torsionally constrained DNA molecule induced by the immobilized helicase activity directly in the x- and y- plane as well as rotation of the DNA molecule in real time, and novel spFRET assays to understand the mechanism of action of a helicase. We will utilize the PcrA helicase in these studies. Helicases play critical roles in DNA and RNA metabolism, including DNA replication, repair, recombination and transcription. The genomes of Gram-positive bacteria encode an essential, conserved helicase, PcrA, which is involved in DNA repair and in plasmid rolling-circle replication, and also in as yet unidentified essential cellular processes. The experiments described in this proposal are likely to lead to the development of new instruments capable of combining the strengths of MT and spFRET, expand the ability of a SCFM to follow double spFRET, arid expand the use of MT to follow longitudinal and rotational movements generated by a motor protein on DNA. These instruments will allow studies on not only DNA helicases but also other motor proteins that act on nucleic acids, such as DNA and RNA polymerases, DNA repair and recombination factors, chromatin remodeling factors, etc. The experiments described here will also increase our understanding of the mechanism of action of an important group of motor proteins as exemplified by the PcrA helicase which is involved in a number of DNA transactions. ? ? ?
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