The uvsY recombination protein of bacteriophage T4 forms hexamers in the presence and absence of single-stranded DNA.
A prerequisite to genetic recombination in the T4 bacteriophage is the formation of the presynaptic filament-a helical nucleoprotein filament containing stoichiometric amounts of the uvsX recombinase in complex with single-stranded DNA (ssDNA). Once formed, the filament is competent to catalyze homologous pairing and DNA strand exchange reactions. An important component in the formation of the presynaptic filament is the uvsY protein, which is required for optimal uvsX-ssDNA assembly in vitro, and essential for phage recombination in vivo. uvsY enhances uvsX activities by promoting filament formation and stabilizing filaments under conditions of low uvsX, high salt, and/or high gp32 (ssDNA-binding protein) concentrations. The molecular properties of uvsY include noncooperative binding to ssDNA and specific protein-protein interactions with both uvsX and gp32. Evidence suggests that all of these hetero-associations of the uvsY protein are important for presynaptic filament formation. However, there is currently no structural information available on the uvsY protein itself. In this study, we present the first characterization of the self-association of uvsY. Using hydrodynamic methods, we demonstrate that uvsY associates into a stable hexamer (s020,w = 6.0, M = 95 kDa) in solution and that this structure is competent to bind ssDNA. We further demonstrate that uvsY hexamers are capable of reversible association into higher aggregates in a manner dependent on both salt and protein concentration. The implications for presynaptic filament formation are discussed.
Beernink
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HT
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405, USA.
Morrical
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SW
D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics; D002498:Centrifugation; D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011995:Recombination, Genetic; D012965:Sodium Chloride; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D008565:Membrane Proteins; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; D012965:Sodium Chloride