Protein-nucleic acid interactions




RecA protein-dsDNA complex imaged by atomic force microscopy (AFM):

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Present research is centered on the biochemical mechanism and biological function of protein-nucleic acid interactions.

Currently the lab is studying proteins (DNA strand exchange proteins; DNA helicases; nucleases; and DNA binding proteins) and DNA intermediates (homologously-paired joint molecules and Holliday junctions) that are involved in the process of genetic recombination. The experimental approaches include molecular genetic (site-specific mutagenesis), biochemical (enzyme kinetics; DNA footprinting; and crosslinking), and biophysical (atomic force microscopy and spectroscopic) techniques.

Equilibrium and kinetic methods are being used to define the determinants of both protein-DNA and homologous DNA-DNA recognition. Atomic force microscopy is being used to determine the structures of these complexes in both the hydrated state and in solution; the latter approach affords the possibility of visualizing reactions at the single-molecular level. Finally, fluorescence and optical trapping methods are being used to visualize the translocation of DNA helicases or "motors" along DNA.

    The laboratory is examining the RecA, RecBCD, and SSB proteins, of E. coli and the Rad51, Rad52, and RP-A proteins of S. cerevisiae. The goals of our work are both to establish in vitro systems that accurately reproduce the cellular process and to understand the biochemical function of each participant in these complex reactions.

    The RecA protein promotes a reaction that is both biochemically unique and central to the recombination process, namely, the ATP-dependent homologous pairing and exchange of DNA strands; despite considerable study, many biochemical aspects of this reaction remain unclear, including the mechanisms of both homologous DNA recognition and energy transduction.

    The RecBCD enzyme is a DNA helicase that is also an ATP-dependent nuclease. It interacts with a specific DNA sequence, essential for elevated recombination activity, referred to as Chi site. Through an unknown biochemical mechanism, Chi sites attenuate the nuclease activity, but not the helicase activity, of RecBCD enzyme.

    The SSB protein is a single-stranded DNA binding protein that stimulates the activities of both the RecA and RecBCD proteins by virtue of its ability to bind ssDNA. Proteins with similar activities have been found in eukaryotes as well.

    The Rad51 protein shows considerable sequence similarity to the RecA protein. Similarly, the RP-A protein is presumably the functional equivalent of the SSB protein. The manner by which the activities of these proteins are coordinated to produce novel product DNA molecules is being examined.

    The experimental approaches used include the thermodynamic, kinetic, and structural techniques. We have used fluorescence, UV, and infrared spectroscopy, atomic force microscopy (AFM), stopped-flow kinetics, spectroscopic and electrophoretic assays for enzymatic activity, protein-nucleic acid crosslinking, chemical modification, electron microscopy, video-enhanced fluorescence microscopy, optical trapping, site-specific mutagenesis, and in vivo assays for protein-protein interactions.