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 accesso ry protein of the phage-encoded replicative DNA helicase, gp41. The pr operties of this 26 kDa protein include selective binding to ssDNA, an d 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 essent ial 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 pr esent the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluore scence enhancement and quantitative ssDNA-cellulose methods, we demons trate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monom er; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > epsilon DNA (random sequence) > poly(dA) > poly(d epsilon A); (3) gp59 exhibits two or more different polynucleotide binding mo des distinguished by their cooperativities of binding, and modulated b y salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with m ultiple ionic contacts between protein and ssDNA phosphate residues an d with the displacement of anions from the protein. The implications o f our findings for the mechanism of action of gp59 in helicase-ssDNA a ssembly are discussed. (C) 1997 Academic Press Limited.