Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase α-primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase δ assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single-stranded DNA-binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition-based protein switches in which two proteins vie for contact with RPA.

This study reports a primase-to-polymerase switch in E. coli that closely links primase action with extension by DNA polymerase III holoenzyme. We find that primase tightly grips its RNA primer, protecting it from the action of other proteins. However, primase must be displaced before the beta sliding clamp can be assembled on the primed site. A single subunit of the holoenzyme, chi, is dedicated to this primase displacement task. The displacement mechanism depends on a third protein, SSB. Primase requires contact to SSB for its grip on the primed site. The chi subunit also binds SSB, upon which the primase-to-SSB contact is destabilized leading to dissociation of primase and assembly of beta onto the RNA primer. The conservation of this three-point switch, in which two proteins exchange places on DNA via mutually exclusive interaction with a third protein, is discussed.

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the β sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called γ complex that uses ATP to assemble the β clamp onto DNA. We examine here the function of the χ subunit of the γ complex clamp loader. Omission of χ from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for χ and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with χ. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to χ. These results establish a role for the χ subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.

The E. coli replicase, DNA polymerase III holoenzyme, contains two polymerases for replication of duplex DNA. The DNA strands are antiparallel requiring different modes of replicating the two strands: one is continuous (leading) while the other is discontinuous (lagging). The two polymerases within holoenzyme are generally thought to have asymmetric functions for replication of these two strands. This report finds that the two polymerases have equal properties, both are capable of replicating the more difficult lagging strand. Asymmetric action is, however, imposed by the helicase that encircles the lagging strand. The helicase contact defines the leading polymerase constraining it to a subset of actions, while leaving the other to cycle on the lagging strand. The symmetric actions of the two polymerases free holoenzyme to assemble into the replisome in either orientation without concern for a correct match to one or the other strand.