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.

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.

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.

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.

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.

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).