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|Title:||Complex Formation between Phage φ29 Single-stranded DNA Binding Protein and DNA|
|Authors:||Soengas, M. S., Esteban, José A., Salas, Margarita, Gutiérrez, C.|
|Abstract:||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.|
|Publisher version (URL):||http://dx.doi.org/10.1006/jmbi.1994.1364|
|Citation:||Journal of Molecular Biology 239(2): 213-226 (1994)|
|Appears in Collections:||(CBM) Artículos|
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