Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

Abstract

DNA replication of phi29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of phi29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of phi29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during phi29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by phi29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the phi29 and Nf SSBs do not show any effect. On the other hand, the phi29 SSB is the only one of the three SSBs able to increase the replication rate of phi29 DNA polymerase in primed M13 ssDNA replication. From the fact that the phi29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of phi29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on phi29-like DNA replication has been proposed.

Figure 1

Sequence alignment of the φ29, Nf and GA-1 SSBs. Identical residues in the three SSBs are white on a black background. Similar residues of the φ29 and/or Nf SSBs with respect to the GA-1 SSB, the least related of them, are black on a grey background. Similarities between the φ29 and Nf SSBs are not highlighted. Numbers indicate the amino acid position.

Figure 2

Helix-destabilizing activity of the φ29, Nf and GA-1 SSBs. An M13 ssDNA molecule to which a 5′-labeled 17mer oligonucleotide was hybridized was incubated with increasing amounts of either the φ29, Nf or GA-1 SSB or the corresponding buffer. After 40 min at 37°C, reactions were stopped and fractionated on a polyacrylamide gel, as indicated in Materials and Methods. Positions of the hybrid substrate and the displaced oligonucleotide are indicated. C is the control of the heat-denatured substrate.

Figure 3

Ability of the φ29, Nf and GA-1 SSBs to prevent non-productive binding of φ29 DNA polymerase to ssDNA. The assays were carried out as described in Materials and Methods, using a 5′-labeled 15mer oligonucleotide as substrate for the 3′→5′ exonucleolytic activity of φ29 DNA polymerase. When the enzyme was preincubated with M13, all DNA polymerases were trapped and no degradation of the oligonucleotide was detected. The indicated amounts of the φ29, Nf or GA-1 SSB were added to the preincubation mixture of φ29 DNA polymerase and M13 ssDNA. After incubation for 75 s at 30°C, degradation of the labeled DNA was analyzed by electrophoresis in 20% polyacrylamide gels in the presence of 8 M urea and autoradiography. Control of each SSB alone, at the highest amount employed in the assay, is also shown in each last lane. Positions of the 15mer oligonucleotide and of the degradation products are indicated.

Figure 4

Dose-dependent effect of the φ29 (A), Nf (B) and GA-1 (C) SSBs on φ29 DNA polymerase in primed M13 ssDNA replication. The assay was carried out as described in Materials and Methods using 25 ng of φ29 DNA polymerase and the indicated amounts of SSB or the corresponding buffer. After incubation at 30°C for the indicated times, dNTP incorporation was calculated by counting the Cèrenkov radiation of the synthesized DNA and the replication rate was measured by analysis of the replication products by alkaline 0.7% agarose gel electrophoresis and autoradiography in the presence of size markers. Two different expositions are shown for the highest protein concentration used in the case of φ29 and Nf SSBs. The size of the newly synthesized DNA was estimated after densitometric scanning of the autoradiograms. Positions of the molecular size markers are indicated.

Figure 5

Graphs of the length of the synthesized DNA versus time and tables with the respective replication rates corresponding to the M13 DNA replication assays shown in Figure 4. (A) Stimulatory effect of increasing amounts of the φ29 SSB on φ29 DNA polymerase. Values corresponding to 0 (open circles), 3.5 (closed triangles), 7 (open triangles), 14 (open squares) and 28 µM (closed circles) φ29 SSB are shown. (B and C) Replication rate of φ29 DNA polymerase in the presence of increasing amounts of the Nf and GA-1 SSBs, respectively. Values corresponding to 0 (open circles) and the respective highest protein concentrations tested (closed circles) (25.2 and 13 µM) are shown, as intermediate concentrations did not display different behavior.

Figure 6

Effect of the φ29 and Nf SSBs on the replication rate of φ29 and Nf DNA polymerases. The assay was carried out as described in Materials and Methods. Primed M13 ssDNA was incubated with 25 µM of the indicated SSB or the corresponding buffer. After addition of 25 ng of φ29 (A) or Nf (B) DNA polymerases, samples were incubated at 30°C for the indicated times and subjected to alkaline 0.7% agarose gel electrophoresis. Positions of the molecular size markers are indicated. Graphs of the length of the synthesized DNA versus time and the tables with the respective replication rates corresponding to both (A) and (B) are also shown. Values corresponding to the DNA synthesized in the absence (open circles) and in the presence of the φ29 (closed circles) and Nf (triangles) SSBs are displayed.

Figure 7

Association–dissociation equilibrium of SSB binding to DNA in type I and II RIs generated during φ29-like DNA replication. Primer TP is indicated in black and parental TP is shaded. DBP p6, dsDNA-binding protein; SSB p5, ssDNA-binding protein. Only the left DNA end has been drawn except for type I and type II molecules, where the two DNA ends are shown.