DSpace

Digital.CSIC > Biología y Biomedicina > Centro de Biología Molecular Severo Ochoa (CBM) > (CBM) Artículos >

Share

EndNote

Impact

Links

Closed Access item Complex Formation between Phage φ29 Single-stranded DNA Binding Protein and DNA

Authors:Soengas, M. S.
Esteban, J. A.
Salas, Margarita
Gutiérrez, C.
Keywords:Nucleoprotein complex, SSB
Issue Date:2-Jun-1994
Publisher:Elsevier
Citation:Journal of Molecular Biology 239(2): 213-226 (1994)
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
URI:http://hdl.handle.net/10261/38856
ISSN:0022-2836
10.1006/jmbi.1994.1364
Appears in Collections:(CBM) Artículos

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.