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Bacteriophage φ29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB) which stimulates viral DNA replication. In the present study, a structural characterization of the complex between ssDNA and the φ29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of φ29 SSB upon binding. Phage φ29 SSB behaves as a monomer in solution and forms complexes with ssDNA which have a homogeneous structure, as if they consist of a continuous array of protein bound to DNA. Interaction of φ29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of φ29 SSB bound to the ssDNA and the maximal quenching upon binding was very high (Qmax = 94?6±3·5%). Direct titration experiments have allowed us to estimate that the stoichiometry (n) of binding to ssDNA was 3·4(±0·3) nucleotides per φ29 SSB monomer. Both Qmax and n are independent of the salt concentration, suggesting the existence of only one major binding mode. At low salt concentrations, the effective binding constant (Keff = Kw) to poly(dT) was 2·2 × 105 M-1 the intrinsic binding constant (K) and the cooperativity parameter (w) being 4·3 × 103 M-1 and 51, respectively. At increasing salt concentrations, the Keff exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of φ29 SSB during viral DNA replication is discussed., This investigation has been aided by Research grant from the National Institutes of Health (5 ROl GM27242-14)

Publisher Summary Topoisomer gel retardation is useful for the study of interactions of anti-Z-DNA antibodies with left-handed Z-DNA, reevaluation of binding specificities displayed by presumptive Z-DNA-binding proteins, study of repressor-mediated DNA looping in the lac and araBAD operons, as well as for study of the influence of DNA supercoiling on such looping phenomena. This chapter describes the basic features of the gel retardation technique and demonstrates some applications. The application discussed are the binding of anti-Z-DNA antibodies (both intact immunoglobulin molecules and Fab fragments) to Z-DNA, the identification of HeLa nuclear extract activities with an apparent affinity for structural discontinuities associated with B-Z junctions, and the association of Escherichia coli single-strand-specific DNA binding protein with negatively supercoiled minicircles containing stretches of Z-DNA.

Publisher Summary This chapter discusses the interactions of proteins with synthesized fragments of DNA by convenient physicochemical and enzymological methods. It defines such a comprehensive approach as an organochemical one. Strict positional directionality of modifications and their multiple uses are an essential factor of this approach. In selecting the type of modification, it aspired to the maximal similarity of the structures of double-stranded oligo(po1y)nucleotides with respect to native fragments of DNA. The conformation of modified compounds is a special question. The organochemical approach became two achievements of laboratory: the synthesis of concatemer DNA duplexes and the development of chemical ligation enabling directed modifications in DNA duplexes. This chapter sums up the data obtained in laboratory in studying, by the organochemical approach, specific and nonspecific DNA­-protein interactions. As a “nonspecific” test protein, gene-5 protein is selected from bacteriophage fl (P5) belonging to the group of single-stranded-DNA-binding (SSB) proteins. These proteins are involved in DNA synthesis in prokaryotes and eukaryotes. They have a higher affinity for single-stranded than for double-stranded DNA and thus destabilize the double-stranded DNA.