Differential functional behavior of viral phi 29, Nf and GA-1 SSB proteins

DNA replication of phi 29 and related phages takes place via a strand displ acement mechanism, a process that generates large amounts of single-strande d DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) ar e essential proteins during phage phi 29-like DNA replication. In the prese nt work we analyze the helix-destabilizing activity of the SSBs of phi 29 a nd the related phages Nf and GA-l,their ability to eliminate non-productive binding of phi 29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi 29 DNA polymerase in primed M13 ssDNA replication, a sit uation that resembles type II replicative intermediates that occur during p hi 29-like DNA replication. Significant differences have been appreciated i n the functional behavior of the three SSBs, First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation b y phi 29 DNA polymerase in the M13 DNA replication assay, even at SSB conce ntrations at which the phi 29 and Nf SSBs do not show any effect, On the ot her hand, the phi 29 SSB is the only one of the three SSBs able to increase the replication rate of phi 29 DNA polymerase in primed M13 ssDNA replicat ion. From the fact that the phi 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behavi ors of the SSBs on stimulation of the replication rate of phi 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein com plexes of the SSBs with the ssDNA rather than to a specific interaction bet ween the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex fo rmation with the functional stimulatory effect of the SSB on phi 29-like DN A replication has been proposed.