Tau Subunit Research Articles

Replisomal coupling between the α-Pol III core and the τ-subunit of the clamp loader complex (CLC) are essential for genomic integrity in E. coli.

Coupling interactions between the alpha (α) subunit of the polymerase III core (α-Pol III core) and the tau (τ) subunit of the clamp loader complex (τ-CLC) are vital for efficient and rapid DNA replication in Escherichia coli (E. coli). Specific and targeted mutations in the C-terminal τ-interaction region of the Pol III α-subunit disrupted efficient coupled rolling circle DNA synthesis in vitro and caused significant genomic defects in CRISPR-Cas9 dnaE edited cell strains. These α-Pol III mutations eliminated the interaction with τ-CLC but retained wild-type polymerase and exonuclease activities. The most severely affected mutant strains, dnaE:Y1119A and dnaE:L1097/8S, had significantly reduced doubling times, reduced fitness, and increased cellular length phenotypes as a result of this targeted decoupling of the replisome and the generation of replication stress. Those strains also showed significant SOS induction from unwound but unreplicated regions within the genome. In support, significant single-stranded DNA (ssDNA) gaps were detected by fluorescence microscopy and quantified by fluorescence activated cytometry using an in situ PLUG assay for those dnaE:mut strains. By comparing the biochemical and genomic consequences of disrupting the τ-CLC - α-Pol III coupling contacts, we have unveiled a more cohesive picture and mechanistic understanding of replisome dynamics and the essential interactions required to maintain overall fitness through a stable genome.

Tau Mediated Coupling Interactions between Pol III Core DNA Synthesis and DnaB Helicase Unwinding

DNA replication is a vital process ensuring the passing down of intact genetic information over generations. DNA replication in the model organism, Escherichia coli (E. coli), has been extensively studied, providing significant insights into the various enzyme mechanisms. However, while the prime functions of individual components of the replisome have been deciphered, the more complex interactions between the components is an active area of study to understand replisome coupling. At the core of the replisome, the DnaB helicase unwinds the double strand DNA which is followed by rapid synthesis of nascent strand by Polymerase III core (Pol III core). It is proposed that the Tau subunit of the clamp loader complex acts as the physical link between both DnaB and Pol III core, leading to a coupling phenomenon between unwinding and synthesis.Using a reconstituted E. coli replisome in vitro, unwinding of DNA substrates with variable lengths and assemblies can be monitored through electrophoresis to better understand the kinetics of coupling. Mutations have been introduced in the exterior surface of DnaB, disrupting its charged interactions with the excluded strand, and promoting more constricted conformation that result in faster unwinding. The unwinding kinetics of wild‐type versus mutant DnaB enzymes on different length substrates are being examined. Further experiments include Pol III synthesis alone or with proposed coupling from the tau‐subunit and can simultaneously examine unwinding and synthesis. Mutations that affect DnaB conformation or disrupt Pol III‐tau interactions will be utilized to assess their relative influences in maintaining a coupled replisome. Conducting in vitro experiments will provide mechanistic insight into the nature of interactions between these replisome components in a coordinated system.Coupling interactions are also being investigated in vivo using precise CRISPR‐Cas9 genome editing. Coupling mutations have been introduced in the dnaX gene encoding the tau‐subunit. These mutant strains exhibit a reduced growth rate when compared to wild‐type strains, indicating that disrupted coupling leads to reduced doubling times. These strains are being further characterized to determine the genomic and cellular consequences of helicase‐polymerase decoupling in vivo. Therefore, a combination of in vitro and in vivotechniques are being used to better understand the importance of coupling during DNA replication to maintain a stable genome.

Interactions Between the E. Coli Sos Response Protein Umud and DNA Polymerase III Alpha Subunit Have Implications for Regulating Replication in Response to DNA Damage

Faithful and efficient DNA replication is critical for the survival of all organisms; however, DNA is constantly subject to damage from endogenous and exogenous sources. Attempts to replicate damaged DNA can result in harmful mutations. The bacterial SOS response system regulates DNA repair and other pathways responsible for the cellular response to DNA damage. Among genes upregulated during the SOS response is the umuD gene, encoding the manager protein UmuD that subsequently undergoes self-cleavage to remove its N-terminal 24 amino acids to form UmuD'. The umuD gene products are involved in managing the cellular response to DNA damage through their interactions with damage-bypass DNA polymerases DNA pol IV and pol V. UmuD and UmuD' also interact with the alpha DNA polymerase and beta processivity clamp subunits of the replicative DNA polymerase III. We probed the interactions between UmuD or UmuD' and the alpha polymerase subunit and determined that the umuD gene products interact with alpha at two sites: the N-terminal PHP domain, which is responsible for interactions with the epsilon proofreading subunit, and the C-terminal domain, which harbors interaction sites for the beta clamp, the tau subunit of the clamp loader, and single-stranded DNA (ssDNA). The C-terminal domain of alpha preferentially binds to full-length UmuD. With FRET experiments, we subsequently showed that UmuD but not UmuD' inhibits the interaction between alpha and beta, suggesting that early in the SOS response to DNA damage, UmuD could displace alpha from the beta processivity clamp. Furthermore, optical tweezers experiments revealed that UmuD specifically inhibits the interaction between alpha and ssDNA. Together, these observations suggest that UmuD plays a key role in regulating the replication fork after DNA damage.

Simultaneous Interaction of E. Coli Single Stranded DNA Binding Protein and Replicativedna Polymerase III Alpha Subunit with Single-Stranded DNA Molecules

The E. coli single stranded DNA (ssDNA) binding (SSB) protein binds to ssDNA in multiple binding modes and regulates DNA replication via protein-protein interactions. DNA polymerases are enzymes responsible for DNA replication. The alpha subunit of the replicative DNA polymerase III (Pol III) is the DNA polymerase subunit of the 10-subunit bacterial replicase, responsible for DNA replication. It has been shown that replication by the Pol III core is inhibited by SSB and can be relieved by the version of the clamp loader that contains the tau subunit, suggesting that DNA polymerase III is inhibited by SSB unless the replisome complex at the replication fork is fully assembled. Previous work suggests that the mechanism of inhibition is a direct competition between SSB and DNA Pol III core for DNA. SSB inhibits replication by Pol III alpha subunit. We have further shown that there is a direct physical interaction between alpha subunit and SSB. Based on these observations, we hypothesize that the inhibition of Pol III alpha subunit by SSB is due to a specific feedback mechanism facilitated by the direct interaction of the two proteins. To test this hypothesis, we have shown Pol III alpha subunit stabilizes SSB binding to ssDNA using single molecule force-spectroscopy. In the absence of Pol III alpha subunit, SSB stabilizes ssDNA below 20 pN and fully dissociates above 20 pN. However, in the presence of Pol III alpha subunit, SSB does not dissociate above 20 pN and the energy required to dissociate SSB from ssDNA is increased by a factor of two. Therefore, the inhibition of Pol III alpha subunit by SSB is due to a specific interaction between the two proteins.

Investigating Hexameric Helicases: Single-Molecule Studies of DnaB and T4 Gp41

Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the E. coli helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., Cell, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., Biophys. J., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases.

DNA Unwinding By DnaB and the DnaB/TAU Complex

The replicative helicase for E. coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. Using multiplexed single-molecule measurements with magnetic tweezers, we investigate the translocation and unwinding activities of DnaB. We find that DnaB's interaction with the ss/dsDNA junction is dependent on the geometry of the DNA substrate and applied force, suggesting that the hexamer interacts with the occluded strand during unwinding. We have also found that the structure of the bound nucleotides within DnaB's central channel is highly compact relative to the contour length of ssDNA, consistent with crystal structures of related hexameric helicases. Finally, in all our experiments, we find high variance in the rates of unwinding as well as frequent pausing, indicating that individual hexamers fluctuate among different conformations with different activities. To investigate DnaB's variable nature, we test the effect on helicase activity of interactions with the tau subunit of the Pol III holoenzyme, which is thought to regulate DnaB's unwinding rate.

Mechanism of polymerase collision release from sliding clamps on the lagging strand

Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the 'collision release' model, the lagging strand polymerase collides with the 5' terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5' terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the tau subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The tau subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA.

Allosteric regulation of the primase (DnaG) activity by the clamp‐loader (τ) in vitro

During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the tau subunit of the clamp-loader that loads the beta clamp and interconnects the core polymerases on the leading and lagging strands. The tau-DnaB-DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the tau subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB-tau interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V tau mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of tau elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native tau protein. Thus changes in the tau-DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of tau are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by tau-interacting components of the replisome through the tau-DnaB-DnaG pathway.

Measurement of dissociation constants of high-molecular weight protein–protein complexes by transferred 15N-relaxation

The use of (15)N-relaxation data for determination of the dissociation constant of a protein-protein complex is proposed for the situation where a (15)N-labeled protein is bound to an unlabeled protein of high molecular weight, and the chemical exchange between bound and free protein is fast on the NMR time scale. The approach is shown to be suitable for estimating dissociation constants in the micromolar to millimolar range, using protein solutions at relatively low concentration. An example is shown for the interaction between two subunits from the Escherichia coli DNA polymerase III complex, involving a (15)N-labeled fragment of the C-terminal domain of the tau subunit (15 kDa) and the unlabeled alpha subunit (130 kDa).

The unstructured C-terminus of the τ subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the α subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τC16. We show that the extreme C-terminal region of τC16 constitutes the site of interaction with α. The τC16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τC16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (KD) of the α−τC16 complex to be ∼260 pM. Competition with immobilized τC16 by τC16 derivatives for binding to α gave values of KD of 7 μM for the α−τC16Δ7 complex. Low-level expression of the genes encoding τC16 and τC16▵7, but not τC16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τC16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τC24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τC16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.

Transcription Factor IIAτIs Associated with Undifferentiated Cells and Its Gene Expression Is Repressed in Primary Neurons at the Chromatin Level In Vivo

The levels of General Transcription Factor (TF) IIA were examined during mammalian brain development and in rat embryo fibroblasts and transformed cell lines. The large TFIIA subunit paralogues alphabeta and tau are largely produced in unsynchronized cell lines, yet only TFIIA alphabeta is observed in a number of differentiated tissue extracts. Steady-state protein levels of the TFIIA tau, alphabeta, and gamma subunits were significantly reduced when human embryonal (ec) and hepatic carcinoma cell lines were stimulated to differentiate with either all-trans-retinoic acid (ATRA) or sodium butyrate. ATRA-treated NT2-ec cells required replating to induce a neuronal phenotype and loss of detectable TFIIA tau and gamma proteins. High levels of TFIIA tau, alphabeta, and gamma and Sp factors were identified in extracts from human fetal and rat embryonic day-18 brains, but not in human and rat adult brain extracts. A high histone H3 Lys9/Lys4 methylation ratio was observed in the TFIIA tau promoter of primary hippocampal neurons from day-18 rat embryos, suggesting that repressive epigenetic marks of chromatin prevent TFIIA tau from being transcribed in neurons. We conclude that TFIIA tau is associated with undifferentiated cells during development, yet is down-regulated at the chromatin level upon cellular differentiation.

DNA Repeat Rearrangements Mediated by DnaK-Dependent Replication Fork Repair

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.

Amino-acid Type Identification in 15N-HSQC Spectra by Combinatorial Selective 15N-labelling

The efficiency of cell-free protein synthesis combined with combinatorial selective 15N-labelling provides a method for the rapid assignment of 15N-HSQC cross-peaks to the 19 different non-proline amino-acid types from five 15N-HSQC spectra. This strategy was explored with two different constructs of the C-terminal domain V of the tau subunit of the Escherichia coli DNA polymerase III holoenzyme, tauC16 and tauC14. Since each of the five 15N-HSQC spectra contained only about one third of the cross-peaks present in uniformly labelled samples, spectral overlap was much reduced. All 15N-HSQC cross-peaks of the backbone amides could be assigned to the correct amino-acid type. Availability of the residue-type information greatly assisted the evaluation of the changes in chemical shifts observed for corresponding residues in tauC16 vs. those in tauC14, and the analysis of the structure and mobility of the C-terminal residues present in tauC16 but not in tauC14.

Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III τ subunit

To investigate the possible role of accessory subunits of Escherichia coli DNA polymerase III holoenzyme (HE) in determining chromosomal replication fidelity, we have investigated the role of the dnaX gene. This gene encodes both the tau and gamma subunits of HE, which play a central role in the organization and functioning of HE at the replication fork. We find that a classical, temperature-sensitive dnaX allele, dnaX36, displays a pronounced mutator effect, characterized by an unusual specificity: preferential enhancement of transversions and -1 frameshifts. The latter occur predominantly at non-run sequences. The dnaX36 defect does not affect the gamma subunit, but produces a tau subunit carrying a missense substitution (E601K) in its C-terminal domain (domain V) that is involved in interaction with the Pol III alpha subunit. A search for new mutators in the dnaX region of the chromosome yielded six additional dnaX mutators, all carrying a specific tau subunit defect. The new mutators displayed phenotypes similar to dnaX36: strong enhancement of transversions and frameshifts and only weak enhancement for transitions. The combined findings suggest that the tau subunit of HE plays an important role in determining the fidelity of the chromosomal replication, specifically in the avoidance of transversions and frameshift mutations.

Reduced initiation frequency from oriC restores viability of a temperature-sensitive Escherichia coli replisome mutant

The dnaX gene of Escherichia coli encodes tau and gamma clamp loader subunits of the replisome. Cells carrying the temperature-sensitive dnaX2016 mutation were induced for the SOS response at non-permissive temperature. The SOS induction most likely resulted from extensive replication fork collapse that exceeded the cells' capacity for restart. Seven mutations in the dnaA gene that partly suppressed the dnaX2016 temperature sensitivity were isolated and characterized. Each of the mutations caused a single amino acid change in domains III and IV of the DnaA protein, where nucleotide binding and DNA binding, respectively, reside. The diversity of dnaA(Sx) mutants obtained indicated that a direct interaction between the DnaA protein and tau or gamma is unlikely and that the mechanism behind suppression is related to DnaA function. All dnaA(Sx) mutant cells were compromised for initiation of DNA replication, and contained fewer active replication forks than their wild-type counterparts. Conceivably, this led to a reduced number of replication fork collapses within each dnaX2016 dnaA(Sx) cell and prevented the SOS response. Lowered availability of wild-type DnaA protein also led to partial suppression of the dnaX2016 mutation, confirming that the dnaA(Sx) mode of suppression is indirect and results from a reduced initiation frequency at oriC.

Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P.

Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the tau subunit of both DNA polymerases III (Pol IIIs). The B.subtilis full-length protein and their mutant derivatives tau(373- 563) (lacking the N-terminal, domains I-III or amino acid residues 1-372) and tau(1-372) (lacking the C-terminal region or amino acids 373-563) have been purified. The tau protein forms tetramers, tau(373- 563) forms dimers, whereas tau(1-372), depending on the ionic strength, forms trimers or tetramers in solution. In the absence of single-stranded (ss) DNA and a nucleotide cofactor, tau interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry. G40P(109-442), lacking the N-terminal amino acid residues 1-108, interacts with the C-terminal moiety of tau. The data indicate that the interaction of G40P with the tau subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.

A Three-domain Structure for the δ Subunit of the DNA Polymerase III Holoenzyme δ Domain III Binds δ′ and Assembles into the DnaX Complex

Using psi-BLAST, we have developed a method for identifying the poorly conserved delta subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting with Escherichia coli delta, leads not only to the identification of delta but also to the DnaX and delta' subunits of the DnaX complex and other AAA(+)-class ATPases. This suggests that, although not an ATPase, delta is related structurally to the other subunits of the DnaX complex that loads the beta sliding clamp processivity factor onto DNA. To test this prediction, we aligned delta sequences with those of delta' and, using the start of delta' Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of delta. This putative delta Domain III could be expressed to high levels, consistent with the prediction that it folds independently. delta Domain III, like Domain III of DnaX and delta', assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of delta with the DnaX subunits. These observations are consistent with a model where two tau subunits and one each of the gamma, delta', and delta subunits mutually interact to form a pentameric functional core for the DnaX complex.

τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains: DOMAIN IV, LOCATED WITHIN THE UNIQUE C TERMINUS OF τ, BINDS THE REPLICATION FORK HELICASE, DnaB

Interaction between the tau subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In the preceding manuscript, we reported the identification of five putative structural domains within the tau subunit (Gao, D., and McHenry, C. (2000) J. Biol. Chem. 275, 4433-4440). As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged tau fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces. Only tau fusion proteins containing domain IV bound DnaB. The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV. Unlike the binding of immobilized tau(4) to the DnaB hexamer, the binding of monomeric domain IV to DnaB(6) was dependent upon the density of immobilized domain IV, indicating that DnaB(6) is bound by more than one tau protomer. This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric tau. These double tethers of the leading and lagging strand polymerases proceeding through the tau-tau link and an additional tau-DnaB link are likely important for the dynamic activities of the replication fork.

τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains: PARTIAL PROTEOLYSIS OF TERMINALLY TAGGED τ TO DETERMINE CANDIDATE DOMAINS AND TO ASSIGN DOMAIN V AS THE α BINDING DOMAIN

The tau subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta' subunit, and the third region is near the C-terminal end of the tau-delta' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau protein. Domains I and II are based on the crystallographic structure of delta' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau proteins lacking specific protease-resistant domains and analyzed their binding to the alpha subunit by surface plasmon resonance. Results from these studies indicated that the alpha binding site of tau lies within its C-terminal 147 residues (domain V).

Characterization of the Unique C Terminus of theEscherichia coli τ DnaX Protein: MONOMERIC C-τ BINDS α AND DnaB AND CAN PARTIALLY REPLACE τ IN RECONSTITUTED REPLICATION FORKS

A contact between the dimeric tau subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In this report, we exploit the OmpT protease to generate C-tau, a protein containing only the unique C-terminal sequences of tau, free of the sequences shared with the alternative gamma frameshifting product of dnaX. We have established that C-tau is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation. Monomeric C-tau binds the alpha catalytic subunit of DNA polymerase III with a 1:1 stoichiometry. C-tau also binds DnaB, revealed by a coupled immunoblotting method. C-tau restores the rapid replication rate of inefficient forks reconstituted with only the gamma dnaX gene product. The acceleration of the DnaB helicase can be observed in the absence of primase, when only leading-strand replication occurs. This indicates that C-tau, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form.