STRUCTURAL FEATURES OF PHI-29 SINGLE-STRANDED DNA-BINDING PROTEIN .2.GLOBAL CONFORMATION OF PHI-29 SINGLE-STRANDED DNA-BINDING PROTEIN ANDTHE EFFECTS OF COMPLEX-FORMATION ON THE PROTEIN AND THE SINGLE-STRANDED-DNA
Ms. Soengas et al., STRUCTURAL FEATURES OF PHI-29 SINGLE-STRANDED DNA-BINDING PROTEIN .2.GLOBAL CONFORMATION OF PHI-29 SINGLE-STRANDED DNA-BINDING PROTEIN ANDTHE EFFECTS OF COMPLEX-FORMATION ON THE PROTEIN AND THE SINGLE-STRANDED-DNA, The Journal of biological chemistry, 272(1), 1997, pp. 303-310
The strand-displacement mechanism of Bacillus subtilis phage phi 29 DN
A replication occurs through replicative intermediates with high amoun
ts 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 hydr
odynamic behavior of phi 29 SSB in solution and (ii) monitored the eff
ect 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 axi
al ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) o
r 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 (1
5 to 50 degrees C), being mainly constituted by beta-sheet elements (s
imilar to 50%, at pH 7). Complex formation with ssDNA, although induci
ng minimal changes on the global conformation of phi 29 SSB, had a cle
ar stabilizing effect against pH and temperature increase of the solut
ion samples. On the other hand, phi 29 SSB binding leads to non-conser
vative changes of the near-UV CD spectra of ssDNA, which are consisten
t with different nearest-neighbor interactions of the nucleotide bases
upon complex formation. The above results will be compared to those r
eported for other SSBs and discussed in terms of the functional roles
of phi 29 SSB.