Two functional domains of the epsilon subunit of DNA polymerase III

The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.     

Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.     

The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function

The mitochondrial replication machinery in human cells includes the DNA polymerase γ holoenzyme and the TWINKLE helicase. Together, these two factors form a processive replication machinery, a replisome, which can use duplex DNA as template to synthesize long stretches of single-stranded DNA. We here address the importance of the smaller, accessory B subunit of DNA polymerase γ and demonstrate that this subunit is absolutely required for replisome function. The duplex DNA binding activity of the B subunit is needed for coordination of POLγ holoenzyme and TWINKLE helicase activities at the mtDNA replication fork. In the absence of proof for direct physical interactions between the components of the mitochondrial replisome, these functional interactions may explain the strict interdependence of TWINKLE and DNA polymerase γ for mitochondrial DNA synthesis. Furthermore, mutations in TWINKLE as well as in the catalytic A and accessory B subunits of the POLγ holoenzyme, may cause autosomal dominant progressive external ophthalmoplegia, a disorder associated with deletions in mitochondrial DNA. The crucial importance of the B subunit for replisome function may help to explain why mutations in these three proteins cause an identical syndrome.     

Overexpression of the catalytic subunit of DNA polymerase gamma results in depletion of mitochondrial DNA in Drosophila melanogaster

The mechanisms involved in the regulation of mitochondrial DNA (mtDNA) replication, a process that is crucial for mitochondrial biogenesis, are not well understood. In this study, we evaluate the role of DNA polymerase gamma (pol gamma), the key enzyme in mtDNA replication, in both Drosophila cell culture and in developing flies. We report that overexpression of the pol gamma catalytic subunit (pol gamma-alpha) in cultured Schneider cells does not alter either the amount of mtDNA or the growth rate of the culture. The polypeptide is properly targeted to mitochondria, yet the large excess of pol gamma-alpha does not interfere with mtDNA replication under these conditions where the endogenous polypeptide is apparently present in amounts that exceed of the demand for its function in the cell. In striking contrast, overexpression of pol gamma-alpha at the same level in transgenic flies interferes with the mtDNA replication process, presumably by altering the mechanism of DNA synthesis, suggesting differential requirements for, and/or regulation of, mtDNA replication in Drosophila cell culture versus the developing organism. Overexpression of pol gamma-alpha in transgenic flies produces a significant depletion of mtDNA that causes a broad variety of phenotypic effects. These alterations range from pupal lethality to moderate morphological abnormalities in adults. depending on the level and temporal pattern of overexpression. Our results demonstrate that although cells may tolerate a variable amount of the pol gamma catalytic subunit under some conditions, its level may be critical in the context of the whole organism.     

The common A467T mutation in the human mitochondrial DNA polymerase (POLG) compromises catalytic efficiency and interaction with the accessory subunit

Among the nearly 50 disease mutations in the gene for the catalytic subunit of human DNA polymerase gamma, POLG, the A467T substitution is the most common and has been found in 0.6% of the Belgian population. The A467T mutation is associated with a wide range of mitochondrial disorders, including Alpers syndrome, juvenile spinocerebellar ataxia-epilepsy syndrome, and progressive external ophthalmoplegia, each with vastly different clinical presentations, tissue specificities, and ages of onset. The A467T mutant enzyme possesses only 4% of wild-type DNA polymerase activity, and the catalytic defect is manifest primarily through a 6-fold reduction in kcat with minimal effect on exonuclease function. Human DNA polymerase gamma (pol gamma) requires association of a 55-kDa accessory subunit for enhanced DNA binding and highly processive DNA synthesis. However, the A467T mutant enzyme failed to interact with and was not stimulated by the accessory subunit, as judged by processivity, heat inactivation, and N-ethylmaleimide protection assays in vitro. Thermolysin digestion and immunoprecipitation experiments further indicate weak association of the subunits for A467T pol gamma. This is the first example of a mutation in POLG that disrupts physical association of the pol gamma subunits. We propose that reduced polymerase activity and loss of accessory subunit interaction are responsible for the depletion and deletion of mitochondrial DNA observed in patients with this POLG mutation.     

Absence of accessory subunit in the DNA polymerase gamma purified from yeast mitochondria

In yeast and animals, replication of the mitochondrial genome is carried out by the DNA polymerase gamma. In mammals this polymerase is composed of a catalytic and an accessory subunit. Yeast DNA polymerase gamma was purified over 6600-fold from mitochondria. The catalytic polypeptide of this enzyme was identified as a 135-kDa protein by a photochemical crosslinking procedure and its native molecular weight was estimated between 120 and 140 kDa by gel filtration and glycerol gradient sedimentation. These results indicate that yeast DNA polymerase gamma contains only one subunit and thus has a different quaternary structure from its counterpart in animals.     

Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit

gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.     

Baculovirus expression reconstitutes Drosophila mitochondrial DNA polymerase.

Drosophila mitochondrial DNA polymerase has been reconstituted and purified from baculovirus-infected insect cells. Baculoviruses encoding full-length and mature forms of the catalytic and accessory subunits were generated and used in single and co-infection studies. Recombinant heterodimeric holoenzyme was reconstituted in both the mitochondria and cytoplasm of Sf9 cells and required the mitochondrial presequences in both subunits. The recombinant holoenzyme contains DNA polymerase and 3'-5' exonuclease that are stimulated substantially by both salt and mitochondrial single-stranded DNA-binding protein. Thus, the recombinant enzyme exhibits biochemical properties indistinguishable from those of the native enzyme from Drosophila embryos. Production of the catalytic subunit alone yielded soluble protein with the chromatographic properties of the heterodimeric holoenzyme. However, the purified catalytic core has a 50-fold lower specific activity. This provides evidence of a critical role for the accessory subunit in the catalytic efficiency of Drosophila mitochondrial DNA polymerase.     

The herpes simplex virus DNA polymerase: Analysis of the functional domains

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597–961, comprising the homologous sequence regions IV–IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3′-to-5′ exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 ± 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3′-to-5′ exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072–1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.     

DNA polymerase-primase from embryos of Drosophila melanogaster. The DNA polymerase subunit

The DNA polymerase-primase from Drosophila melanogaster has been separated into its constituent polymerase and primase subunits by sedimentation in glycerol gradients containing 50% ethylene glycol. Both activities have been obtained in good yield. The properties of the 182-kDa polymerase subunit are similar to those of the intact four-subunit enzyme. However, there are three significant differences. (i) The polymerase activity of the 182-kDa subunit shows an increased thermolability; (ii) the pause sites during replication of singly primed, single-stranded circular DNA by the 182-kDa subunit are altered; and (iii) unlike the intact enzyme, the 182-kDa subunit is highly processive in the presence of the single-stranded DNA-binding protein of Escherichia coli.     

The influence of the DNA polymerase gamma accessory subunit on base excision repair by the catalytic subunit

Mammalian DNA polymerase gamma, the sole polymerase responsible for replication and repair of mitochondrial DNA, contains a large catalytic subunit and a smaller accessory subunit, pol gammaB. In addition to the polymerase domain, the large subunit contains a 3'-5' editing exonuclease domain as well as a dRP lyase activity that can remove a 5'-deoxyribosephosphate (dRP) group during base excision repair. We show that the accessory subunit enhances the ability of the catalytic subunit to function in base excision repair mainly by stimulating two subreactions in the repair process. Pol gammaB appeared to specifically enhance the rate at which pol gamma was able to locate damage in high molecular weight DNA. One pol gammaB point mutant known to have impaired ability to bind duplex DNA stimulated repair poorly, suggesting that duplex DNA binding through pol gammaB may help the catalytic subunit locate sites of DNA damage. In addition, the small subunit significantly stimulated the dRP lyase activity of pol gammaA, although it did not increase the rate at which the dRP group dissociated from the enzyme. The ability of DNA pol gamma to process a high load of damaged DNA may be compromised by the slow release of the dRP group.     

A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.     

Multiple forms of rat liver mitochondrial DNA polymerase.

Mitochondrial DNA polymerase (DNA polymerase mt) exists in two active forms. DNA polymerase present in crude extract (M-I) and ammonium sulfate precipitate (M-II) stages of purification sediments at 12.1S. The enzyme at the M-II stage of purification has a molecular weight of approximately 250,000 as determined by Sephadex G-200 chromatography in buffers of low ionic strength. In buffers containing 0.15 m NaCl, the enzyme sediments at 9.4S and has a molecular weight of approximately 190,000. When the enzyme is further purified on diethylaminoethyl cellulose (M-III stage of purification), the 9.4S activity predominates. Addition of a polymerase-free fraction from the M-III stage of purification changes the sedimentation coefficient of the enzyme from 9.4 to 12.1S.     

A mitochondrial DNA polymerase from embryos of Drosophila melanogaster. Purification, subunit structure, and partial characterization

The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a Stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase gamma is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phi X174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase gamma as partially purified from several vertebrates.     

The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.     

Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases

The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.     

Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit.

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.