Complex formation between phage phi 29 single-stranded DNA binding protein and DNA.
Bacteriophage phi 29 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 phi 29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of phi 29 SSB upon binding. Phage phi 29 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 phi 29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of phi 29 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 phi 29 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 = K omega) to poly(dT) was 2.2 x 10(5) M-1, the intrinsic binding constant (K) and the cooperativity parameter (omega) being 4.3 x 10(3) 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 phi 29 SSB during viral DNA replication is discussed.
Soengas
M S
MS
Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Universidad Autónoma, Cantoblanco, Madrid, Spain.
Esteban
J A
JA
Salas
M
M
Gutiérrez
C
C
D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D005814:Genes, Viral; D007700:Kinetics; D008854:Microscopy, Electron; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins