Simultaneous Interaction of E. Coli Single Stranded DNA Binding Protein and Replicativedna Polymerase III Alpha Subunit with Single-Stranded DNA Molecules

The E. coli single stranded DNA (ssDNA) binding (SSB) protein binds to ssDNA in multiple binding modes and regulates DNA replication via protein-protein interactions. DNA polymerases are enzymes responsible for DNA replication. The alpha subunit of the replicative DNA polymerase III (Pol III) is the DNA polymerase subunit of the 10-subunit bacterial replicase, responsible for DNA replication. It has been shown that replication by the Pol III core is inhibited by SSB and can be relieved by the version of the clamp loader that contains the tau subunit, suggesting that DNA polymerase III is inhibited by SSB unless the replisome complex at the replication fork is fully assembled. Previous work suggests that the mechanism of inhibition is a direct competition between SSB and DNA Pol III core for DNA. SSB inhibits replication by Pol III alpha subunit. We have further shown that there is a direct physical interaction between alpha subunit and SSB. Based on these observations, we hypothesize that the inhibition of Pol III alpha subunit by SSB is due to a specific feedback mechanism facilitated by the direct interaction of the two proteins. To test this hypothesis, we have shown Pol III alpha subunit stabilizes SSB binding to ssDNA using single molecule force-spectroscopy. In the absence of Pol III alpha subunit, SSB stabilizes ssDNA below 20 pN and fully dissociates above 20 pN. However, in the presence of Pol III alpha subunit, SSB does not dissociate above 20 pN and the energy required to dissociate SSB from ssDNA is increased by a factor of two. Therefore, the inhibition of Pol III alpha subunit by SSB is due to a specific interaction between the two proteins.