Summary:

The biochemistry behind DNA polymerase III dissociation from DNA during Okazaki fragment synthesis (the “lagging-strand processivity switch”) has been investigated to the greatest degree in Escherichia coli. Currently, it is understood that the t subunit of the DNA polymerase III holoenzyme facilitates dissociation of the clamp processivity factor ( b subunit) and the a subunit (polymerase), which allows a to bind a new primed site for replication. Furthermore, it has recently been shown that t is responsible for sensing a single-strand nick, which is presented as the penultimate step in the synthesis of an Okazaki fragment. However, the structural basis for t’s ability to dissociate a from b is currently unknown. The goal of my project is to utilize X-ray crystallography to investigate the inner workings of the processivity switch in the replisome of the thermophilic bacterium Thermus thermophilus. Below is a cartoon taken from Johnson and O'Donnell, 2005 which depicts a model by which the t subunit is thought to dissociate the clamp processivity factor from the polymerase:
 

As the cartoon suggests, upon encountering a nicked DNA substrate, the t subunit outcompetes the clamp processivity factor for the C-terminal tail of the polymerase. However, past evidence from the laboratory of Charles McHenry suggests that the polymerase actually contains another polymerase site that has been recently shown to be a higher affinity binding site for the clamp processivity factor. The existence and nature of this second binding site adds new complexity to the dissociation mechanism and increases the need for structural answers.

Commentary on this work:

Dohrmann, P.R., and McHenry, C.S. (2005) A Bipartite Polymerase-Processivity Factor Interaction: Only the Internal b Binding Site of the a Subunit is Required for Processive Replication by the DNA Polymerase III Holoenzyme J. Mol. Bio., 350, 228 – 239.

Johnson, A. and O'Donnell, M. (2005) Cellular DNA Replicases: Components and Dynamics at the Replication Fork. Annu. Rev. Biochem. 74: 283 - 315.

Kim, D. R., and McHenry, C.S. (1996) Identification of the beta-binding domain of the alpha subunit of Escherichia coli polymerase III holoenzyme. J. Biol. Chem. 271(34): 20699 - 704.

Leu, F. P., Georgescu, R. E., and O’Donnell, M. (2003) Mechanism of the E. coli t Processivity Switch during Lagging-Strand Synthesis. Mol. Cell, 11: 315 – 327.