Inhibition of purified recombinant reverse transcriptase from wild-type and zidovudine-resistant clinical isolates of human immunodeficiency virus type 1 by zidovudine, stavudine, and lamivudine triphosphates.

Cross-resistance between zidovudine, stavudine, and lamivudine was studied, using purified recombinant reverse transcriptase from a zidovudine-susceptible and -resistant pair of clinical isolates of human immunodeficiency virus type 1. The zidovudine-resistant isolate exhibited low-level cross-resistance to both stavudine and lamivudine in drug susceptibility assays. Enzyme from the resistant isolate demonstrated reduced inhibition by zidovudine triphosphate and stavudine triphosphate and, to a lesser extent, lamivudine triphosphate. These findings provide additional evidence at the viral and enzyme level for cross-resistance between zidovudine and stavudine, and they suggest a possible effect of zidovudine resistance on susceptibility to lamivudine.

Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly.

The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template. The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains. The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta'. In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels. The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III. Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex. A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.

Carboxyl-terminal domain III of the delta' subunit of the DNA polymerase III holoenzyme binds delta.

The delta and delta' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2gammadeltadelta'chipsi). The x-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both NH2- and COOH-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. delta and delta' form a 1:1 heterodimer with a dissociation constant (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) determined by surface plasmon resonance was comparable. Domain III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.

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.

Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core.

Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.

tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX.

The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433-4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.

The delta and delta ' subunits of the DNA polymerase III holoenzyme are essential for initiation complex formation and processive elongation.

delta and delta' are required for assembly of the processivity factor beta(2) onto primed DNA in the DNA polymerase III holoenzyme-catalyzed reaction. We developed protocols for generating highly purified preparations of delta and delta'. In holoenzyme reconstitution assays, delta' could not be replaced by delta, tau, or gamma, even when either of the latter were present at a 10,000-fold molar excess. Likewise, delta could not be replaced by delta', tau, or gamma. Bacterial strains bearing chromosomal knockouts of either the holA(delta) or holB(delta') genes were not viable, demonstrating that both delta and delta' are essential. Western blots of isolated initiation complexes demonstrated the presence of both delta and delta'. However, in the absence of chipsi and single-stranded DNA-binding protein, a stable initiation complex lacking deltadelta' was isolated by gel filtration. Lack of delta-delta' decreased the rate of elongation about 3-fold, and the extent of processive replication was significantly decreased. Adding back delta-delta' but not chipsi, delta, or delta' alone restored the diminished activity, indicating that in addition to being key components required for the beta loading activity of the DnaX complex, deltadelta' is present in initiation complex and is required for processive elongation.

Tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain III, shared by gamma and tau, binds delta delta ' and chi psi.

The DnaX complex of the DNA polymerase holoenzyme assembles the beta(2) processivity factor onto the primed template enabling highly processive replication. The key ATPases within this complex are tau and gamma, alternative frameshift products of the dnaX gene. Of the five domains of tau, I-III are shared with gamma In vivo, gamma binds the auxiliary subunits deltadelta' and chipsi (Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017-3020). To localize deltadelta' and chipsi binding domains within gamma domains I-III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to deltadelta' and chipsi by surface plasmon resonance. Fusion proteins containing either DnaX domains I-III or domains III-V bound deltadelta' and chipsi subunits. A DnaX protein only containing domains I and II did not bind deltadelta' or chipsi. The binding affinity of chipsi for DnaX domains I-III and domains III-V was the same as that of chipsi for full-length tau, indicating that domain III contained all structural elements required for chipsi binding. Domain III of tau also contained deltadelta' binding sites, although the interaction between deltadelta' and domains III-V of tau was 10-fold weaker than the interaction between deltadelta' and full length tau. The presence of both delta and chipsi strengthened the delta'-C(0)tau interaction by at least 15-fold. Domain III was the only domain common to all of tau fusion proteins whose interaction with delta' was enhanced in the presence of delta and chipsi. Thus, domain III of the DnaX proteins not only contains the deltadelta' and chipsi binding sites but also contains the elements required for the positive cooperative assembly of the DnaX complex.

tau binds and organizes Escherichia coli replication through distinct domains. Partial proteolysis of terminally tagged tau to determine candidate domains and to assign domain V as the alpha 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).

tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain IV, located within the unique C terminus of tau, 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.

A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of deltadelta' with DnaX(4) forms DnaX(3)deltadelta'.

We have constructed a plasmid-borne artificial operon that expresses the six subunits of the DnaX complex of Escherichia coli DNA polymerase III holoenzyme: tau, gamma, delta, delta', chi and psi. Induction of this operon followed by assembly in vivo produced two taugamma mixed DnaX complexes with stoichiometries of tau(1)gamma(2)deltadelta'chipsi and tau(2)gamma(1)deltadelta'chipsi rather than the expected gamma(2)tau(2)deltadelta'chipsi. We observed the same heterogeneity when taugamma mixed DnaX complexes were reconstituted in vitro. Re-examination of homomeric DnaX tau and gamma complexes assembled either in vitro or in vivo also revealed a stoichiometry of DnaX(3)deltadelta'chipsi. 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.

Characterization of the unique C terminus of the Escherichia coli tau DnaX protein. Monomeric C-tau binds alpha AND DnaB and can partially replace tau 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.

The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme.

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction. delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348-13357). Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme.

Identification of the acidic residues in the active site of DNA polymerase III.

The mechanism of nucleotide addition by DNA polymerases involves two metal ions that are coordinated in the active site by conserved acidic residues. The three acidic residues that chelate Mg2+ in the active site of Escherichia coli DNA polymerase III have been identified as Asp401, Asp403, and Asp555 by site-directed mutagenesis. Candidates for mutagenesis were initially chosen based on absolute conservation of acidic residues in an alignment of more than 20 diverse DnaE sequences. Conservative Asp to Glu mutations at positions 401 and 403 reduced the activities of the mutant polymerases 2000 and 333-fold, respectively, from that of the wild-type. The third carboxylate was identified by a series of mutations for each critical candidate. With the exception of Glu, all of the mutations at Asp555 led to severely diminished polymerase activity, while each of the other candidates exhibited several relatively active mutant polymerases. Moreover, only the identified active site mutant polymerases displayed a significant enhancement of activity in Mn2+ compared with Mg2+. These data suggest a direct involvement of the mutated amino acid in metal ion binding.

Mismatch extension by Escherichia coli DNA polymerase III holoenzyme.

The in vitro fidelity of Escherichia coli DNA polymerase III holoenzyme (HE) is characterized by an unusual propensity for generating (-1)-frameshift mutations. Here we have examined the capability of HE isolated from both a wild-type and a proofreading-impaired mutD5 strain to polymerize from M13mp2 DNA primer-templates containing a terminal T(template).C mismatch. These substrates contained either an A or a G as the next (5') template base. The assay allows distinction between: (i) direct extension of the terminal C (producing a base substitution), (ii) exonucleolytic removal of the C, or (iii), for the G-containing template, extension after misalignment of the C on the next template G (producing a (-1)-frameshift). On the A-containing substrate, both HEs did not extend the terminal C (<1%); instead, they exonucleolytically removed it (>99%). In contrast, on the G-containing substrate, the MutD5 HE yielded 61% (-1)-frameshifts and 6% base substitutions. The wild-type HE mostly excised the mispaired C from this substrate before extension (98%), but among the 2% mutants, (-1)-frameshifts exceeded base substitutions by 20 to 1. The preference of polymerase III HE for misalignment extension over direct mismatch extension provides a basis for explaining the in vitro (-1)-frameshift specificity of polymerase III HE.

The chi psi 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 chi and psi 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 chipsi-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-tau-psi chi. gamma, the alternative product of the dnaX gene, will not replace tau in this reaction, indicating that tau's unique ability to bind to DNA polymerase III holding chipsi in the same complex is essential. All of our findings are consistent with chipsi strengthening DNA polymerase III holoenzyme interactions with the SSB-coated lagging strand at the replication fork, facilitating complex assembly and elongation.

The base substitution and frameshift fidelity of Escherichia coli DNA polymerase III holoenzyme in vitro.

We have investigated the in vitro fidelity 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 lacId forward mutational target. The error rate for both enzymes was lowest at low dNTP concentrations (10-50 microM) and highest at high dNTP concentration (1000 microM). 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.

Role of the core DNA polymerase III subunits at the replication fork. Alpha is the only subunit required for processive replication.

The DNA polymerase III holoenzyme is composed of 10 subunits. The core of the polymerase contains the catalytic polymerase subunit, alpha, the proofreading 3'-->5' exonuclease, epsilon, and a subunit of unknown function, theta. The availability of the holoenzyme subunits in purified form has allowed us to investigate their roles at the replication fork. We show here that of the three subunits in the core polymerase, only alpha is required to form processive replication forks that move at high rates and that exhibit coupled leading- and lagging-strand synthesis in vitro. Taken together with previous data this suggests that the primary determinant of replication fork processivity is the interaction between another holoenzyme subunit, tau, and the replication fork helicase, DnaB.

Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme.

Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.

A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium.

We have purified a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the alpha catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the gamma and tau subunits of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene. The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria. Examination of the sequence permitted identification of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter gamma DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this finding are discussed.