Structural features of phi 29 single-stranded DNA-binding protein .2. Global conformation of phi 29 single-stranded DNA-binding protein and the effects of complex formation on the protein and the single-stranded DNA

The strand-displacement mechanism of Bacillus subtilis phage phi 29 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, phi 29 SSB, to be replicated in vivo. To understand the characteristics of phi 29 SSB-ssDNA complex that could explain the requirement of phi 29 SSB, we have (i) determined the hydrodynamic behavior of phi 29 SSB in solution and (ii) monitored the effect of complex formation on phi 29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi 29 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 phi 29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (similar to 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi 29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi 29 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 phi 29 SSB.