The broad goal of this research program is to elucidate the molecular mechanisms by which enzyme complexes on single-stranded DNA are assembled and activated. The interaction of specific enzymes with ssDNA is an essential feature of the mechanisms of DNA replication. recombination, and repair that occur in all living cells. The assembly and activation of enzyme-ssDNA complexes is complicated, however, by the presence of helix- destabilizing proteins (HDPs) that bind tightly and cooperatively to ssDNA. To bind to ssDNA and activate its catalytic activities, an enzyme must first displace HDPs, an energetically unfavorable process. Therefore special HDP displacement mechanisms appear to be required to load an enzyme onto a preexisting HDP-ssDNA matrix. We propose to study these displacement and loading mechanisms by investigating the molecular steps involved in the assembly and activation of two different model enzyme- ssDNA complexes: that of a DNA helicase, the bacteriophage T4 gene 41 protein (gp41), and that of a general recombinase, the T4 uvsX protein. The assembly and activation of each enzyme-ssDNA complex, appears to require the protein-ssDNA and/or protein-protein interactions of as many as three different phage proteins: the enzyme itself, an enzyme-specific assembly factor, and a specific HDP-- the T4 gene 32 protein (gp32). Our specific objective is therefore to dissect the protein-ssDNA and protein- protein interactions involved in the loading of gp41 and uvsX onto gp32- ssDNA. This will be accomplished by analyzing the interactions of the two assembly factors with ssDNA, with gp32 and with their enzyme partners. Protein-ssDNA interactions will be examined directly by fluorescence spectroscopy and indirectly by coupled spectrophotometric assay. Protein- protein interactions will be examined by a combination of protein affinity chromatography and sedimentation methods and by the isolation and characterization of specifically altered proteins. It is expected that these studies will identify the important macromolecular interactions required for the assembly and activation of enzyme-ssDNA complexes, and for the displacement of HDPs from ssDNA by other proteins. In a more general sense, identifying the macromolecular interactions required for the displacement of one protein by another on ssDNA should contribute to our understanding of many other protein displacement reactions that occur during the various genetic processes, including the displacement of nucleosomes by transcription factors and the displacement of one protein by another during the processing and intracellular transport of messenger RNAs.
