Assembly and Activation of Enzyme-ssDNA Complexes

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DESCRIPTION (provided by applicant): The objective of this research program is to characterize the roles of "mediator proteins" in assembling recombinase and helicase enzymes onto single-stranded DNA. Our model is the DNA replication/recombination system of bacteriophage 14. We will study the assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA. We will also study the assembly of gp41, the essential DNA helicase component of the T4 primosome, onto ssDNA. Both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. In both cases, proper assembly requires the activity of a specific mediator protein (uvsY or gp59, respectively). This project focuses on the mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. Our approach is biochemical, and will utilize solution methods including fluorescence, sedimentation, and affinity chromatography, plus physical methods including X-ray crystallography and electron microscopy. Our SPECIFIC AIMS are the following: AIM #1: Investigate the mechanism of T4 presynaptic filament assembly. We will test the following hypotheses: (A) That binding of uvsY hexamers to gp32-ssDNA disrupts gp32 cooperativity, facilitating the displacement of gp32 by uvsX protein: and (B) That binding of uvsY hexamers stabilizes a high-affinity ssDNA-binding conformation of uvsX, facilitating nucleation of uvsX-ssDNA filaments. AIM #2: Perform X-ray crystallography studies of uvsY, the T4 recombination mediator protein. The atomic structure of uvsY will be solved alone and in complex with bound ssDNA and/or derivatives of gp32 protein. AIM #3: Investigate the mechanism of helicase loading by T4 gp59 protein. We will test the following specific hypotheses: (A) That gp59 forms discrete clusters on gp32-ssDNA which remodel the complex and facilitate the displacement of gp32 by gp41 helicase; (B) That binding of gp32's "A-domain" destabilizes gp59-DNA interactions by altering the conformation of gp59's HMG-Iike "N-domain"; and (C) That gp59 promotes gp41 ring-hexamer formation by inducing or stabilizing a high-affinity NIP binding conformation of the helicase.