COMPLEX-FORMATION BETWEEN PHAGE PHI-29 SINGLE-STRANDED-DNA BINDING-PROTEIN AND DNA

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 (Q(max) = 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 Q(max) 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 (K(eff) = K(ω)) to poly(dT) was 2.2 × 105 M−1, the intrinsic binding constant (K) and the cooperativity parameter (φ) being 4·3 × 103 M−1 and 51, respectively. At increasing salt concentrations, the K(eff) exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of φ29 SSB during viral DNA replication is discussed. © 1994 Academic Press, Inc.