DNA replication results in duplication of chromosomes before cell division. Due to the antiparallel nature of DNA, one of the strands (leading strand) is copied continuously, whereas the other (lagging strand) is replicated discontinuously and requires repetitive RNA priming by DnaG primase. A three point switch model was proposed in 1999 to describe the recycling of primase on lagging strands. It involves three interactions, starting from that between DnaB helicase and DnaG to lay down an RNA primer. After primer synthesis, DnaG dissociates from DnaB but remains bound to the primer by contacting single-stranded DNA-binding (SSB) protein on the lagging-strand loop. Finally, DnaG is displaced from SSB by interaction with the chi subunit of DNA polymerase III holoenzyme, releasing the primer to DNA polymerase for DNA synthesis.

In this work, we identified the residues required for interaction between DnaB and the C-terminal domain of DnaG (DnaGC) by viability screening of mutants. Secondly, the interacting domains between DnaG and SSB were pinpointed to DnaGC and the last 8 residues of SSB (SSB-Ct), and the crystal structure of the DnaGC:SSB-Ct complex was determined. Biophysical studies revealed that although the binding sites on DnaGC for DnaB and SSB are distinct, binding of DnaB and SSB-Ct to DnaGC are mutually exclusive. This reveals an allosteric switch on DnaGC where binding to SSB promotes dissociation of DnaB, explaining one aspect of the mechanism of the three point switch. Finally, the crystal structure of chi in complex with the SSB-Ct was determined. Comparison of the structures of SSB-Ct in complex with DnaGC and chi revealed a similar conformation, different from those of SSB-Ct in complex with other proteins (ExoI and RecO). The similar conformation of SSB-Ct in complex with DnaGC and chi make these interactions good targets for antibiotics directed at disruption of lagging-strand DNA synthesis.