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

Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.

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

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-C(tag)) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except ß) from this strain contained one molecule of γ-C(tag) 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 α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known ß-binding domain, decreasing interaction with the ß2 processivity factor. Surprisingly, mutations within the ß binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized.

The rate of polymerase release upon filling the gap between Okazaki fragments is inadequate to support cycling during lagging strand synthesis.

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 s to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5'-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 min. Reduction in gap size to 1 nt increased the rate of dissociation 2.5-fold, and complete filling of the gap increased the off-rate an additional 3-fold (t(1/2)~2 min). An exogenous primed template and ATP accelerated dissociation an additional 4-fold in a reaction that required complete filling of the gap. Neither a 5'-triphosphate nor a 5'-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nt) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-cross-linking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating that it serves as the sensor.

Novel role for checkpoint Rad53 protein kinase in the initiation of chromosomal DNA replication in Saccharomyces cerevisiae.

A novel role for Rad53 in the initiation of DNA replication that is independent of checkpoint or deoxynucleotide regulation is proposed. Rad53 kinase is part of a signal transduction pathway involved in the DNA damage and replication checkpoints, while Cdc7-Dbf4 kinase (DDK) is important for the initiation of DNA replication. In addition to the known cdc7-rad53 synthetic lethality, rad53 mutations suppress mcm5-bob1, a mutation in the replicative MCM helicase that bypasses DDK's essential role. Rad53 kinase activity but neither checkpoint FHA domain is required. Conversely, Rad53 kinase can be activated without DDK. Rad53's role in replication is independent of both DNA and mitotic checkpoints because mutations in other checkpoint genes that act upstream or downstream of RAD53 or in the mitotic checkpoint do not exhibit these phenotypes. Because Rad53 binds an origin of replication mainly through its kinase domain and rad53 null mutants display a minichromosome loss phenotype, Rad53 is important in the initiation of DNA replication, as are DDK and Mcm2-7 proteins. This unique requirement for Rad53 can be suppressed by the deletion of the major histone H3/H4 gene pair, indicating that Rad53 may be regulating initiation by controlling histone protein levels and/or by affecting origin chromatin structure.

A bipartite polymerase-processivity factor interaction: only the internal beta binding site of the alpha subunit is required for processive replication by the DNA polymerase III holoenzyme.

Previously, we localized the beta2 interacting portion of the catalytic subunit (alpha) of DNA polymerase III to the C-terminal half, downstream of the polymerase active site. Since then, two different beta2 binding sites within this region have been proposed. An internal site includes amino acid residues 920-924 (QADMF) and an extreme C-terminal site includes amino acid residues 1154-1159 (QVELEF). To permit determination of their relative contributions, we made mutations in both sites and evaluated the biochemical, genetic, and protein binding properties of the mutant alpha subunits. All purified mutant alpha subunits retained near wild-type polymerase function, which was measured in non-processive gap-filling assays. Mutations in the internal site abolished the ability of mutant alpha subunits to participate in processive synthesis. Replacement of the five-residue internal sequence with AAAKK eliminated detectable binding to beta2. In addition, mutation of residues required for beta2 binding abolished the ability of the resulting polymerase to participate in chromosomal replication in vivo. In contrast, mutations in the C-terminal site exhibited near wild-type phenotypes. alpha Subunits with the C-terminal site completely removed could participate in processive DNA replication, could bind beta2, and, if induced to high level expression, could complement a temperature-sensitive conditional lethal dnaE mutation. C-terminal defects that only partially complemented correlated with a defect in binding to tau, not beta2. A C-terminal deletion only reduced beta2 binding fourfold; tau binding was decreased ca 400-fold. The context in which the beta2 binding site was presented made an enormous difference. Replacement of the internal site with a consensus beta2 binding sequence increased the affinity of the resulting alpha for beta2 over 100-fold, whereas the same modification at the C-terminal site did not significantly increase binding. The implications of multiple interactions between a replicase and its processivity factor, including applications to polymerase cycling and interchange with other polymerases and factors at the replication fork, are discussed.