The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

Abstract

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.

DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks

“…That the size of fragments is identical regardless of content suggests that all three forms of Pol III HE experience the same dynamics at the replication fork, initiating and cycling between Okazaki fragment synthesis at nearly equivalent rates, resulting in the same length of gaps between fragments. It has been proposed that 3 Pol III HE leaves shorter gaps between Okazaki fragments than Pol III HE containing two s. These experiments were performed by a low-resolution singlemolecule technique where a single pixel covered 600 nucleotides, approaching the size of the 1-to 2-kb Okazaki fragments found under physiological conditions. To surmount this obstacle, conditions were altered to increase Okazaki fragment size to 12 kb (21).…”

“…Recently, we have established that the major cellular form of Pol III HE is Pol III 2 2 ␥␦␦', disproving proposals that the cellular replicase contains three s and three polymerases (3). One of the observations that was initially used to support the three-polymerase model was that three Pol III assemblies synthesized DNA in a coupled fork system with smaller gaps between Okazaki fragments (21).…”

“…Cells that cannot express ␥ exhibit defects in UV viability and Pol IV-mediated mutagenesis (3). A proposal has been made that ␥ may be required to avoid the presence of a third Pol III at the replication fork that might outcompete other polymerases, such as Pol IV, that must enter the replication fork to resolve unrepairable lesions and to perform stress-induced mutagenesis functions (2,3).…”

Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

“…The PolC-mCherry foci likely represent the site(s) of active DNA replication (2). Photobleaching experiments and in vitro measurements of E. coli have found that three copies of DnaE are positioned at each replication fork (7,30), while other in vitro studies have found two copies (9). It is therefore important to determine the stoichiometry of the essential DNA polymerase PolC in B. subtilis.…”

“…It is therefore appropriate that great efforts have been made to better understand how DNA polymerases function both in vitro and in vivo. For example, the stoichiometry and architecture of the replicative DNA polymerase holoenzyme (Pol III) have been well characterized in the model organism Escherichia coli (7)(8)(9). Although E. coli has served as a prototype for understanding DNA synthesis in vivo, and although some E. coli DNA replication features are conserved across species, the replisomes of many other bacterial species have distinct organizations and operate differently (10)(11)(12).…”