| The prokaryotic replisome is the protein assembly that accomplishes DNA replication in bacteria. The DnaB helicase forms the core of the replisome along with the alpha (PolIIIalpha) and tau (PolIIItau) subunits of the DNA Polymerase III holoenzyme. PolIIIalpha, a C-family DNA polymerase, is the catalytic subunit of the PolIII holoenzyme and carries out DNA synthesis. PolIIPtau triggers polymerase cycling on the lagging strand and acts as the glue that holds the replication fork together through its interactions with both the DnaB helicase and PolIIIalpha. These proteins provide a system to study how a cohesive and processive machine can also function dynamically.;The crystal structure of unliganded PolIIIalpha from Thermus aquaticus revealed that the polymerase utilizes a polbeta-like nucleotidyltransferase (betaNT) fold in its palm domain as do members of the X family of DNA polymerases such as the eukaryotic repair polymerase Polbeta. This finding demonstrated that the replicative polymerases of prokaryotes and eukaryotes evolved separately since eukaryotic replicative DNA polymerases utilize a classic palm fold. Additionally, the structures of the PHP-nuclease active site, an internal binding site for the clamp, and a potential pre-insertion nucleotide binding site within the fingers were determined.;The crystal structure of a ternary complex of PolIIIalpha from T. aquaticus bound to primer-template DNA and incoming nucleotide revealed that, contrary to a competing model within the field, PolIIIalpha does indeed bind DNA as Polbeta does. The OB-fold was observed to undergo a conformational change to bind to the downstream template DNA that emerges from the polymerase active site of PolIIIalpha. Finally, using a structure of the clamp bound to DNA, a model of PolIIIalpha bound to the clamp demonstrated that adequate space existed for two polymerases to simultaneously bind the clamp and supported a model of polymerase switching on the clamp.;Recent work focused on understanding how PolIIItau triggers the lagging-strand processivity switch and interacts with the DnaB helicase. Crystals of PolIIItau and DnaB that diffract to 8 A were isolated. Electron density maps generated from molecular replacement phases suggest that PolIIItau may bind to the central channel or between the two rings of the DnaB hexamer. |
