Investigation of protein-protein interactions of single-stranded DNA-binding proteins by analytical ultracentrifugation.

Bacterial single-stranded DNA-binding (SSB) proteins are essential for DNA metabolism, since they protect stretches of single-stranded DNA and are required for numerous crucial protein-protein interactions in DNA replication, recombination, and repair. At the lagging strand of the DNA replication fork of Escherichia coli, for example, SSB contacts not only DnaG primase but also the χ subunit of DNA polymerase III, thereby facilitating the switch between primase and polymerase activity. Here, we describe a powerful method that allows the study of interactions between SSB and its binding partners by sedimentation velocity experiments in an analytical ultracentrifuge. Whenever two molecules interact, a complex of a higher mass forms that can usually be distinguished from free binding partners by its different sedimentation behavior. As an example, we show how sedimentation velocity experiments of purified proteins can be employed to determine the binding parameters of the interaction of SSB and the χ subunit of DNA polymerase III from E. coli.