Charles S. McHenry

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.

Chromosomal replicases as asymmetric dimers: studies of subunit arrangement and functional consequences

Studies of the DNA polymerase III holoenzyme of Escherichia coli support a model in which both the leading and lagging strand polymerases are held together in a complex with the replicative helicase and priming activities, allowing two identical α catalytic subunits to assume different functions on the two strands of the replication fork. Creation of distinct functions for each of the two polymerases within the holoenzyme depends on the asymmetric character of the entire complex. The asymmetry of the holoenzyme is created by the DnaX complex, a heptamer that includes τ and γ products of the dnaX gene. τ and γ perform unique functions in the DnaX complex, and the interaction between α and τ appears to dictate the catalytic subunits role in the replicative reaction. This review considers the properties of the DnaX complex including both τ and γ, with the goal of understanding the properties of the replicase and its function in vivo. Recent studies in eukaryotic and other prokaryotic systems suggest that an asymmetric dimeric replicase may be universal. The leading and lagging strand polymerases may be distinct in some systems. For example, Pol e and Pol δ may function as distinct leading and lagging strand polymerases in eukaryotes, and PolC and DnaE may function as distinct leading and lagging strand polymerases in low GC content Gram‐positive bacteria.

The χψ Subunits of DNA Polymerase III Holoenzyme Bind to Single-stranded DNA-binding Protein (SSB) and Facilitate Replication of an SSB-coated Template

A complex of the χ and ψ proteins is required to confer resistance to high levels of glutamate on the DNA polymerase III holoenzyme-catalyzed reaction (Olson, M., Dallmann, H. G., and McHenry, C. (1995) J. Biol. Chem. 270, 29570–29577). We demonstrate that this salt resistance also requires templates to be coated with the Escherichia coli single-stranded DNA-binding protein (SSB). We show that this is the result of a direct χψ–SSB interaction that is strengthened approximately 1000-fold when SSB is bound to DNA. On model oligonucleotide templates, DNA polymerase III core is inhibited by SSB. We show that the minimal polymerase assembly that will synthesize DNA on SSB-coated templates is polymerase III–τ–ψχ. γ, the alternative product of thednaX gene, will not replace τ in this reaction, indicating that τ’s unique ability to bind to DNA polymerase III holding χψ in the same complex is essential. All of our findings are consistent with χψ strengthening DNA polymerase III holoenzyme interactions with the SSB-coated lagging strand at the replication fork, facilitating complex assembly and elongation.

DNA replicases from a bacterial perspective.

Bacterial replicases are complex, tripartite replicative machines. They contain a polymerase, polymerase III (Pol III), a β₂ processivity factor, and a DnaX complex ATPase that loads β₂ onto DNA and chaperones Pol III onto the newly loaded β₂. Bacterial replicases are highly processive, yet cycle rapidly during Okazaki fragment synthesis in a regulated way. Many bacteria encode both a full-length τ and a shorter γ form of DnaX by a variety of mechanisms. γ appears to be uniquely placed in a single position relative to two τ protomers in a pentameric ring. The polymerase catalytic subunit of Pol III, α, contains a PHP domain that not only binds to a prototypical ε Mg²⁺-dependent exonuclease, but also contains a second Zn²⁺-dependent proofreading exonuclease, at least in some bacteria. This review focuses on a critical evaluation of recent literature and concepts pertaining to the above issues and suggests specific areas that require further investigation.

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.

The DNA Polymerase III Holoenzyme: An Asymmetric Dimeric Replicative Complex with Leading and Lagging Strand Polymerases

The DNA Polymerase III holoenzyme forms initiation complexes on primed DNA in an ATP-dependent reaction. We demonstrate that the nonhydrolyzable ATP analog, ATP gamma S, supports the formation of an isolable leading strand complex that loads and replicates the lagging strand only in the presence of ATP, beta, and the single-stranded DNA binding protein. The single endogenous DnaX complex within DNA polymerase III holoenzyme assembles beta onto both the leading and lagging strand polymerases by an ordered mechanism. The dimeric replication complex disassembles in the opposite order from which it assembled. Upon ATP gamma S-induced dissociation, the leading strand polymerase is refractory to disassembly allowing cycling to occur exclusively on the lagging strand. These results establish holoenzyme as an intrinsic asymmetric dimer with distinguishable leading and lagging strand polymerases.

Preparation of extracts from prokaryotes

Publisher Summary Enzymatic lysis methods minimize denaturation, are scale independent, and allow some selectivity in the release of cellular products. The drawbacks to enzymatic methods include the large number of variables that can influence lysis and the addition of substances that may complicate subsequent purification steps. Degradation of the peptidoglycan in gram-negative cells is made more difficult by the presence of an asymmetric lipid bilayer. The outer membrane is external to the peptidoglycan and acts as a permeability barrier to large molecules. Thus, gram-negative bacteria are less susceptible than gram-positive bacteria to lysozyme and detergents. The factors influencing the efficiency of lysis include rate of agitation, cell concentration, concentration of glass beads, diameter of the beads, residence time in the chamber, and temperature. All these factors may need to be determined empirically. Sonication lyses cells by liquid shear and cavitation. Sonication remains a popular technique for lysing small quantities of cells, but is of limited value for cell quantities in the 50-g to 1-kg range because of the difficulty in maintaining low temperatures.

Biotin Tagging Deletion Analysis of Domain Limits Involved in Protein-Macromolecular Interactions MAPPING THE τ BINDING DOMAIN OF THE DNA POLYMERASE III α SUBUNIT

The τ subunit dimerizes DNA polymerase III via interaction with the α subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the α subunit were constructed and purified by Ni2+-nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each α deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the τ binding activity of each α deletion was analyzed using surface plasmon resonance. The τ subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with α (KD ∼ 70 pM). Four additional NH2-terminal α deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the τ subunit (KD = 0.19-0.39 nM), whereas deletion of 705 residues or more from the NH2 terminus of the α subunit abolished τ binding activity. Full-length α that contained a carboxyl-terminal fusion with the biotinylation sequence bound τ strongly (KD = 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated τ binding. These results indicate that the COOH-terminal half of the α subunit is involved in τ interaction.

The Base Substitution and Frameshift Fidelity of Escherichia coli DNA Polymerase III Holoenzyme in Vitro

We have investigated the in vitrofidelity of Escherichia coli DNA polymerase III holoenzyme from a wild-type and a proofreading-impaired mutD5 strain. Exonuclease assays showed the mutD5 holoenzyme to have a 30–50-fold reduced 3′→5′-exonuclease activity. Fidelity was assayed during gap-filling synthesis across the lacI dforward mutational target. The error rate for both enzymes was lowest at low dNTP concentrations (10–50 μM) and highest at high dNTP concentration (1000 μM). The mutD5 proofreading defect increased the error rate by only 3–5-fold. Both enzymes produced a high level of (−1)-frameshift mutations in addition to base substitutions. The base substitutions were mainly C→T, G→T, and G→C, but dNTP pool imbalances suggested that these may reflect misincorporations opposite damaged template bases and that, instead, T→C, G→A, and C→T transitions represent the normal polymerase III-mediated base·base mispairs. The frequent (−1)-frameshift mutations do not result from direct slippage but may be generated via a mechanism involving “misincorporation plus slippage.” Measurements of the fidelity of wild-type and mutD5 holoenzyme during M13 in vivo replication revealed significant differences between the in vivo and in vitro fidelity with regard to both the frequency of frameshift errors and the extent of proofreading.

A coproofreading Zn 2+ -dependent exonuclease within a bacterial replicase

The proofreading exonucleases of all DNA replicases contain acidic residues that chelate two Mg2+ ions that participate in catalysis. DNA polymerase III holoenzymes contain their proofreading activity in a separate subunit, ε, which binds the polymerase subunit, α, through αs N-terminal php domain. Here we demonstrate that the α php domain contains a novel Zn2+-dependent 3′ → 5′ exonuclease that preferentially removes mispaired nucleotides, providing the first example of a coediting nuclease.