The strand-displacement mechanism of Bacillus subtilis phage 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, 29 SSB, to be replicated in vivo. To understand the characteristics of 29 SSBssDNA complex that could explain the requirement of 29 SSB, we have (i) determined the hydrodynamic behavior of 29 SSB in solution and (ii) monitored the effect of complex formation on 29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient In all organisms studied so far, key cellular processes related to DNA metabolism, such as DNA replication, DNA repair, DNA recombination, or DNA transcription, occur via transient ssDNA 1 intermediates whose structure must be properly organized. This organization is achieved and maintained by highly efficient, localized (sequence-specific), or generalized (sequence-independent) protein contacts. Noncatalytic proteins that bind ssDNA with a relatively high affinity in a sequence independent manner have been referred to as single-stranded DNA-binding proteins, SSBs, (1-4). One of the most striking features of the SSBs is the lack of any clear structural common characteristics among them. Thus, the oligomeric state of these proteins in solution is highly variable. They have being found as monomers, e.g. N4 SSB (5); 29 SSB (6); dimers, e.g. SSBs from filamentous Fd phages (3); T7 gene 2.5 (7); tetramers, e.g. Escherichia coli SSB (8); multioligomers, T4 gp32 (1); or heterooligomers, e.g. human SSB (9) and Drosophila SSB (10). Moreover, although several attempts to find structural motifs in SSBs have been made (11,12), only among SSBs of very related filamentous phages a limited sequence homology has been found within the so-called DNA binding wing (see Ref. 3, for review). NMR and crystallographic studies showed a similar three-dimensional structure of the SSBs of E. coli and Pseudomonas aeruginosa filamentous phages Ff and Pf3 (13-15), rather different from that of T4 gp32, the other SSB 2 whose structure (x-ray difraction) has been reported (18). In addition, detailed analysis of a few SSB-ssDNA complexes indicate that each protein interacts with ssDNA using a particular set of residues (3, 15, 19 -21) and induces different conformational changes in the DNA structure (see Refs. 2,4,and 8, for reviews). The functional characteristics of the SSBs complicate even more this variability. Most of the SSBs activate DNA replication, e.g. E. coli SSB, T4 gp32, T7 gene 2.5 protein, 29 SSB, and human SSB (reviewed in Ref. 4). However, other SSBs, as those of the filamentous phages, block DNA replication (22). As a consequence of this diversity, the molecular nature of the SSB-ssDNA interactions is not yet well understood.The requirement for a SSB is particularly important during strand-displacement DNA replication, where great amounts of ssDNA are produced. This ssDNA corresponds to the strand that is not used as template and is displaced as the DN...