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Structural Features of Φ 29 Single-stranded DNA-binding Protein: II. global conformation of Φ 29 single-stranded dna-binding protein and the effects of complex formation on the protein and the single-stranded DNA

AutorSoenga, María S.; Reyes Mateo, C.; Rivas, Germán ; Salas, Margarita; Acuña, A. Ulises ; Gutiérrez Armenta, Crisanto
Palabras claveBacillus subtilis
Fecha de publicación3-ene-1997
EditorAmerican Society for Biochemistry and Molecular Biology
CitaciónJournal of Biological Chemistry 272(1): 303-310 (1997)
ResumenThe strand-displacement mechanism of Bacillus subtilis phage Φ29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, Φ29 SSB, to be replicated in vivo. To understand the characteristics of Φ29 SSB-ssDNA complex that could explain the requirement of Φ29 SSB, we have (i) determined the hydrodynamic behavior of Φ29 SSB in solution and (ii) monitored the effect of complex formation on Φ29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 ± 0.1) × 108 gs1, and its rotational correlation time, 7.0 ± 0.5 ns, Φ29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that 29 SSB is highly organized within a wide range of temperatures (15 to 50 °C), being mainly constituted by -sheet elements (~50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of Φ29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, Φ29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of Φ29 SSB.
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