Probing Conformational Transition Pathways of Protein-DNA Complexes by Polarization-Modulated Fourier Imaging Correlation Spectroscopy (PM-FICS)

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This award in the Chemistry of Life Processes (CLP) program supports work by Professor Andrew H. Marcus at the University of Oregon to carry out fundamental studies on the molecular mechanisms of DNA polymerase and helicase, two protein-DNA complexes that are essential elements of the biological processes that enact and regulate gene expression. Pairs of fluorescent nucleic acid base analogues are substituted for native nucleotides at the forks and junctions of double stranded and primer-template DNA. Polarized fluorescence fluctuation measurements of the naked DNA constructs will reveal information about the relative dipole angle of the base analogue sites, and their relative motions on microsecond to sub-second time scales. Subsequent experiments will be carried out on protein-DNA complexes to directly probe the mechanics of the protein enzymatic sites during key steps of the replication process. These studies will provide detailed information about the transition state pathways of DNA synthesis, and investigate the possible existence of intermediate states involved in replication fidelity. A significant component of the research involves active recruitment and mentoring of students from under-represented areas into the undergraduate and graduate training programs. These studies will add to our basic understanding of the dynamic processes whereby the macromolecular machines of gene expression operate in performing their biological functions. Potential benefits range from an improved understanding of the molecular level interface between chemical and biological behavior, to new general insights about the underlying principles of nano-machines.