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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.

The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages 29 and Nf. In this work, the selfinteraction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo crosslinking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of 29 and Nf. Three deletion mutant proteins have been characterized, ⌬N19, ⌬N26, and ⌬N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein ⌬N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins ⌬N26 and ⌬N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.Single-stranded DNA-binding proteins (SSBs) 1 contribute to DNA metabolism, playing essential roles in different processes such as DNA replication, repair, and recombination (1-3). SSBs bind single-stranded DNA (ssDNA) in a selective, cooperative, and non-sequence-specific way, protecting it from nuclease attack and preventing the formation of secondary structures on it. SSBs are ubiquitous. They have been isolated from bacteria and their phages, eukarya and their viruses, and archaea (1-6).Different oligomerization states have been reported for SSBs. Thus, monomeric (T4gp32 from bacteriophage T4 or AdDBP from adenovirus (5)), dimeric (SSBs from filamentous phages M13 (1) and Pf3 (7)), homotetrameric (EcoSSB from Escherichia coli (2, 8) or human mitochondrial SSB (9)), and heterotrimeric (hRPA, human RPA (3)) SSBs have been described. Important differences have been also reported mainly concerning the amino acid sequence and ssDNA binding properties of the proteins of this family. The function of the SSB is particularly important in the case of organisms that replicate their genetic material via a proteinpriming mechanism followed by strand displacement, as large amounts of ssDNA are generated in the process (10 -12). This is the case of adenovirus and of the 29 family of Bacillus phages. The genome of the latter consists of a linear dsDNA molecule of about 20 kb that contains a phage-encoded terminal protein (TP) covalently linked to each 5Ј end. Replication of the viral genome starts at either DNA end non-simultaneously by a protein-priming mechanism. After a sliding-back step (13), the viral DNA polymerase elongates the initiation product proceeding by strand displacement toward the other DNA end. The displaced strand is cooperatively bound by the viral...