ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES

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DESCRIPTION (adapted from investigator's abstract): The objective of Dr. Morrical's research is to characterize the protein-ssDNA and protein-protein interactions required for the assembly and activation of enzyme complexes on single-stranded DNA. Two different representative enzyme-ssDNA assembly problems in the bacteriophage T4 DNA recombination and replication systems will be examined: (1) Assembly of presynaptic filaments containing the T4 uvsX recombinase bound to ssDNA; and (2) Assembly of gp41, the essential DNA helicase component of the phage primosome, onto ssDNA. A common feature is that both enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound gp32, the T4 ssDNA-binding protein. Another common feature is that both enzymes require a second "assembly factor" (uvsY or gp59, respectively) for assembly onto gp32-ssDNA complexes. This project focuses on the detailed biochemical mechanisms used by uvsY to load uvsX, and by gp59 to load gp41, onto gp32-covered ssDNA molecules. The SPECIFIC AIMS are the following: AIM 1 Characterize structural and biochemical aspects of uvsY protein and uvsY-ssDNA complexes. The following specific hypotheses will be tested: that ssDNA binds to a hexameric form of uvsY and causes a significant structural rearrangement of the uvsY hexamer; and that uvsY binding causes extensive unstacking of ssDNA bases consistent with wrapping or other structural distortions of the ssDNA. AIM 2 Characterize interactions of uvsY protein with gp32-2ssDNA and uvsX-ssDNA complexes. The following specific hypotheses will be tested: that uvsY retains its hexameric form in complexes with gp32 and uvsX proteins; and that uvsY binding destabilizes gp32-ssDNA interactions while stabilizing uvsX-ssDNA interactions. AIM 3 Characterize interactions of gp59 protein with gp32-ssDNA complexes. The following specific hypotheses will be tested: that a critical cluster of gp59 molecules bound to gp32-ssDNA forms the minimal assembly site for the gp41 helicase; and that gp59 destabilizes gp32-ssDNA complexes through protein-protein interactions with ssDNA-bound gp32 molecules. AIM # 4 Characterize interactions of gp59 protein with the gp41 DNA helicase. The specific hypothesis that gp59 promotes the hexamerization of gp41 will be tested.