Interactions of the bacteriophage T4 gene 59 protein with single-stranded polynucleotides: binding parameters and ion effects.
The gene 59 protein (gp59) of bacteriophage T4 is an important accessory protein of the phage-encoded replicative DNA helicase, gp41. The properties of this 26 kDa protein include selective binding to ssDNA, and specific interactions with both gp41 and gp32, the T4-encoded ssDNA- binding protein. gp59 stimulates many of the DNA-dependent activities of the gp41 enzyme by promoting its assembly onto gp32-ssDNA complexes. Direct interactions between gp59 and gp32-ssDNA complexes are essential for helicase assembly, and gp59-gp32 protein-protein interactions have been shown to play a central role. Presumably, the ssDNA-binding activity of gp59 is also important for helicase assembly; however, to date this activity has been poorly characterized. In this study, we present the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluorescence enhancement and quantitative ssDNA-cellulose methods, we demonstrate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monomer; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > epsilonDNA (random sequence) > poly(dA) > poly(depsilonA); (3) gp59 exhibits two or more different polynucleotide binding modes distinguished by their cooperativities of binding, and modulated by salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with multiple ionic contacts between protein and ssDNA phosphate residues and with the displacement of anions from the protein. The implications of our findings for the mechanism of action of gp59 in helicase-ssDNA assembly are discussed.
Lefebvre
S D
SD
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Morrical
S W
SW
D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics; D017122:Bacteriophage T4; D001665:Binding Sites; D001667:Binding, Competitive; D002845:Chromatography / Q000379:methods; D004265:DNA Helicases / Q000378:metabolism; 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*; D008956:Models, Chemical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012492:Salts; D013050:Spectrometry, Fluorescence; D017186:Titrimetry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012492:Salts; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; D004265:DNA Helicases