Biochemical interactions within a ternary complex of the bacteriophage T4 recombination proteins uvsY and gp32 bound to single-stranded DNA.
The presynaptic phase of homologous recombination requires the formation of a filament of single-stranded DNA (ssDNA) coated with a recombinase enzyme. In bacteriophage T4, at least three proteins are required for the assembly of this presynaptic filament. In addition to the T4 recombinase, uvsX protein, the T4 ssDNA binding protein (gp32), and the uvsY recombination accessory protein are also required. Here we report on a detailed analysis of a tripartite filament containing ssDNA bound by stoichiometric quantities of both uvsY and gp32, which appears to be an important intermediate in the assembly of the T4 presynaptic filament. We demonstrate that uvsY and gp32 simultaneously co-occupy the ssDNA in a noncompetitive fashion. In addition, we show that protein-protein interactions between uvsY and gp32 are not required for the assembly of this ternary complex and do not affect the affinity of uvsY for the ssDNA lattice. Finally, we demonstrate that the interaction of gp32 with the ssDNA is destabilized within this complex, in a manner which is independent of gp32-uvsY interactions. The data suggest that the uvsY protein acts to remodel the gp32-ssDNA complex via uvsY-ssDNA interactions. The implications of these findings for the mechanism of presynapsis in the T4 recombination system are discussed.
Sweezy
M A
MA
Department of Biochemistry, Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405, USA.
Morrical
S W
SW
D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D011485:Protein Binding / Q000235:genetics; D011995:Recombination, Genetic; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; C092864:UvsY protein, Enterobacteria phage T4; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4