Suchergebnisse

DNA replication of ϕ29 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 ϕ29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of ϕ29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of ϕ29 DNA polymerase to ssDNA and their stimulatory effect on replication by ϕ29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during ϕ29-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 ϕ29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the ϕ29 and Nf SSBs do not show any effect. On the other hand, the ϕ29 SSB is the only one of the three SSBs able to increase the replication rate of ϕ29 DNA polymerase in primed M13 ssDNA replication. From the fact that the ϕ29 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 ϕ29 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 ϕ29-like DNA replication has been proposed. ; This investigation was aided by research grant 2RO1 GM27242-20 from the National Institutes of Health, by grant PB93-0173 from the Dirección General de Investigación Científica y Técnica, by grant ERBFMRX CT970115 from the European Union and by an institutional grant from the Fundación Ramón Areces. I.G. was holder of a pre-doctoral fellowship from the Fundación Ramón Areces. ; Peer reviewed