H. Garry Dallmann

Coupling of a Replicative Polymerase and Helicase: A τ–DnaB Interaction Mediates Rapid Replication Fork Movement

The E. coli replication fork synthesizes DNA at the rate of nearly 1000 nt/s. We show here that an interaction between the tau subunit of the replicative polymerase (the DNA polymerase III holoenzyme) and the replication fork DNA helicase (DnaB) is required to mediate this high rate of replication fork movement. In the absence of this interaction, the polymerase follows behind the helicase at a rate equal to the slow (approximately 35 nt/s) unwinding rate of the helicase alone, whereas upon establishing a tau-DnaB contact, DnaB becomes a more effective helicase, increasing its translocation rate by more than 10-fold. This finding establishes the existence of both a physical and communications link between the two major replication machines in the replisome: the DNA polymerase and the primosome.

A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of δδ′ with DnaX4 forms DnaX3δδ′

We have constructed a plasmid‐borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: τ, γ, δ, δ′, χ and ψ. Induction of this operon followed by assembly in vivo produced two τγ mixed DnaX complexes with stoichiometries of τ1γ2δδ′χψ and τ2γ1δδ′χψ rather than the expected γ2τ2δδ′χψ. We observed the same heterogeneity when τγ mixed DnaX complexes were reconstituted in vitro. Re‐examination of homomeric DnaX τ and γ complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX3δδ′χψ. Equilibrium sedimentation analysis showed that free DnaX is a tetramer in equilibrium with a free monomer. An assembly mechanism, in which the association of heterologous subunits with a homomeric complex alters the stoichiometry of the homomeric assembly, is without precedent. The significance of our findings to the architecture of the holoenzyme and the clamp‐assembly apparatus of all other organisms is discussed.

High-throughput screening AlphaScreen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1.

Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27Kip1 expression could improve prognosis and prevent excessive cell proliferation. SCFSkp2 is one of the major ubiquitin E3 ligases responsible for degradation of p27Kip1. Ubiquitination of p27Kip1 also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27Kip1. It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27Kip1 degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27Kip1 ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27Kip1 ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.

Parallel Multiplicative Target Screening against Divergent Bacterial Replicases: Identification of Specific Inhibitors with Broad Spectrum Potential †

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.

Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors of bacterial DNA polymerase III

High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication results in bacterial cell death. The synthesis, structure-activity relationships and functional activity are described.

Discovery and Characterization of the Cryptic Ψ Subunit of the Pseudomonad DNA Replicase

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of α and ϵ (polymerase and editing nuclease), β (processivity factor), and the essential τ, δ, and δ′ components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, χ and Ψ are tightly associated clamp loader accessory subunits. The addition of E. coli χΨ to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of χ and Ψ counterparts in P. aeruginosa. The P. aeruginosa χ subunit was recognizable from sequence similarity, but Ψ was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Ψ subunit, encoded by holD. P. aeruginosa χ and Ψ were co-expressed and purified as a 1:1 complex. P. aeruginosa χΨ increased the specific activity of τ3δδ′ 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between χΨ and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Ψ allowed us to identify Ψ subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Ψ family and confirms the existence of Ψ in several organisms in which Ψ was not identifiable based on sequence similarity alone.

Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

The DnaX complex (DnaX3δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β2, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β2 binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β2. DNA synthesis activity can be restored by increased concentrations of β2. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β2 loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.