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PubMed2005

Consequences of mutations in human DNA polymerase gamma.

DNA polymerase gamma is responsible for replication and repair of the mitochondrial genome. Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a 55-kDa accessory subunit. Mutations in the gene for the catalytic subunit (POLG) have been shown to be a frequent cause of mitochondrial disorders. To date over 40 disease mutations and 9 nonsynonymous polymorphisms in POLG have been found to be associated with autosomal recessive and dominant progressive external ophthalmoplegia (PEO), Alpers syndrome, sensory ataxia, neuropathy, dysarthria and ophthalmoparesis (SANDO), Parkinsonism, and male infertility. In this paper we review the literature of POLG mutations and discuss their impact on mitochondrial diseases. We also describe a public access web database to annotate POLG mutations for the research community.

Amino Acid Sequence↗

PubMed1990

Coprecipitation of 3'----5' exonuclease and DNA polymerase gamma with anti-DNA polymerase gamma antibody.

Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.

Animals↗

PubMed1999

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.

Base Sequence↗

PubMed2000

Characterization of a catalytically slow AP lyase activity in DNA polymerase gamma and other family A DNA polymerases.

Mitochondrial DNA polymerase gamma (pol gamma) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5' side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol gamma acts to catalyze the removal of a 5'-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol gamma is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH(4). For both DNA pol gamma and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol beta, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA.

Base Sequence↗

PubMed1980

Catalysis of adenovirus DNA synthesis in vitro by DNA polymerase gamma.

DNA replication complexes were purified from adenovirus type 5 (Ad5)-infected HeLa cells. DNA synthesis by these complexes in vitro was extremely sensitive to the inhibitors dideoxythymidine triphosphate, N-ethyl maleimide and p-hydroxymercuribenzoate. The bound DNA polymerase was released from the complexes by limited digestion with micrococcal nuclease. This released polymerase preferred poly(rA):(dT)12-18 as template over activated calf thymus DNA. These results are compatible with the major polymerase in the replication complex being of the gamma class.

Adenoviridae↗

PubMed1996

The gamma subfamily of DNA polymerases: cloning of a developmentally regulated cDNA encoding Xenopus laevis mitochondrial DNA polymerase gamma.

We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.

Amino Acid Sequence↗

PubMed2003

DNA polymerase gamma in mitochondrial DNA replication and repair.

Mutations in mitochondrial DNA (mtDNA) are associated with aging, and they can cause tissue degeneration and neuromuscular pathologies known as mitochondrial diseases. Because DNA polymerase gamma (pol gamma) is the enzyme responsible for replication and repair of mitochondrial DNA, the burden of faithful duplication of mitochondrial DNA, both in preventing spontaneous errors and in DNA repair synthesis, falls on pol gamma. Investigating the biological functions of pol gamma and its inhibitors aids our understanding of the sources of mtDNA mutations. In animal cells, pol gamma is composed of two subunits, a larger catalytic subunit of 125-140 kDa and second subunit of 35-55 kDa. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease activity, and a 5'-dRP lyase activity. The accessory subunit is required for highly processive DNA synthesis and increases the affinity of pol gamma to the DNA.

Aging↗

PubMed2006

Inhibitory effect of coenzyme Q on eukaryotic DNA polymerase gamma and DNA topoisomerase II activities on the growth of a human cancer cell line.

Coenzyme Q (CoQ) is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ having shorter isoprenoid chains, especially CoQ1 and CoQ2, selectively inhibited the in vitro activity of eukaryotic DNA polymerase (pol) gamma, which is a mitochondrial pol. These compounds did not influence the activities of nuclear DNA replicative pols such as alpha, delta and epsilon, and nuclear DNA repair-related pols such as beta, eta, iota, kappa and lambda. CoQ also inhibited DNA topoisomerase II (topo II) activity, although the enzymatic characteristics, including modes of action, amino acid sequences and three-dimensional structures, were markedly different from those of pol gamma. These compounds did not inhibit the activities of procaryotic pols such as Escherichia coli pol I, and other DNA metabolic enzymes such as human immunodeficiency virus reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. CoQ1, which has the shortest isoprenoid chains, had the strongest inhibitory effect on pol gamma and topo II activities among CoQ1-CoQ10, with 50% inhibitory concentration (IC50) values of 12.2 and 15.5 microM, respectively. CoQ1 could prevent the growth of human promyelocytic leukemia cells, HL-60, and the 50% lethal dose (LD50) value was 14.0 microM. The cells were halted at S phase and G1 phase in the cell cycle, and suppressed mitochondrial proliferation. From these results, the relationship between the inhibition of pol gamma/topo II and cancer cell growth by CoQ is discussed.

Apoptosis↗

PubMed1999

Sensitive assay for mitochondrial DNA polymerase gamma.

BACKGROUND: The mitochondrial DNA polymerase gamma is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase gamma may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. METHODS: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting. RESULTS: Kinetic analysis of DNA polymerase gamma by this method showed a K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30-30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/microgram, reflecting extensive biologic variation among patients with neuromuscular disease. CONCLUSION: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.

Animals↗

PubMed1981

Identification of a polypeptide component of mouse myeloma DNA polymerase gamma.

Mouse myeloma DNA polymerase gamma was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)n.(dT)12-18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of Mr = 47 000 changed proportionally with DNA polymerase gamma activity. A minor polypeptide of Mr = 140 000 also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by 125I-labeled peptide mapping indicates that the Mr 47 000 polypeptide in the mouse myeloma DNA polymerase gamma preparation is structurally related to the Mr 47 000 polypeptide of chick embryo DNA polymerase gamma (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002-7009). An antibody against chick embryo DNA polymerase gamma cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the Mr 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the Mr 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 per nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the 'expected' specific activity with respect to the Mr 47 000 polypeptide and the molecular weight (Mr = 188 000 on the assumption of a tetrameric structure of the Mr 47 000 polypeptide). Results indicate that the Mr 47 000 polypeptide is a component of the mouse myeloma DNA polymerase gamma.

Animals↗

PubMed1999

The accessory subunit of Xenopus laevis mitochondrial DNA polymerase gamma increases processivity of the catalytic subunit of human DNA polymerase gamma and is related to class II aminoacyl-tRNA synthetases.

Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.

Amino Acid Sequence↗

PubMed1996

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma.

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.

Animals↗

PubMed1981

Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

Adenoviruses, Human↗

PubMed2005

Oxidative stress-induced retinal damage up-regulates DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase in photoreceptor synaptic mitochondria.

Bright light triggers biphasic photoreceptor nuclear DNA fragmentation, suggesting a DNA-repair response (Invest Ophthalmol Vis Sci 43:3511; 2002; Adv Med Biol 533:229-240; Mol Neurobiol 28:111-122). Here, we demonstrate a remarkable increase in expression of the mitochondrial DNA-repair enzymes, DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase, following bright light treatment in rats. DNA polymerase gamma and 8-oxoguanine, the product of guanine oxidation, were selectively localized in photoreceptor synaptic terminals only within the superior central retinal region, where most light damage occurred. All induced DNA polymerase gamma was localized in photoreceptor synaptic terminals after 5 hr of light exposure, despite the fact that most photoreceptor cell mitochondria are confined to the inner segments. The neuroprotective platelet-activating factor-receptor antagonist LAU-0901 decreased mitochondrial DNA polymerase gamma up-regulation, suggesting that its neuroprotective effect is exerted upstream from this event. During aging, the ability to repair damaged photoreceptor DNA greatly declines. Thus, DNA-repair enzymes such as polymerase gamma and 8-oxoguanine-DNA-glycosylase may provide novel pharmacologic targets to promote DNA repair and rescue photoreceptors in retinal degenerative diseases.

Aging↗

PubMed1996

Cloning and characterization of the human mitochondrial DNA polymerase, DNA polymerase gamma.

The nuclear-encoded DNA polymerase gamma (DNA POL gamma) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POL gamma and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POL gamma gene from Drosophila melanogaster and a partial cDNA for DNA POL gamma from Gallus gallus have been cloned. The predicted human DNA POL gamma polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those of Schizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster, and the C-terminal half of G. gallus, respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POL gamma like activity from mitochondrial extracts. The human DNA POL gamma is unique in that the first exon of the gene contains a CAG10 trinucleotide repeat.

Amino Acid Sequence↗

PubMed2004

DNA polymerase gamma, the mitochondrial replicase.

DNA polymerase (pol) gamma is the sole DNA polymerase in animal mitochondria. Biochemical and genetic evidence document a key role for pol gamma in mitochondrial DNA replication, and whereas DNA repair and recombination were thought to be limited or absent in animal mitochondria, both have been demonstrated in recent years. Thus, the mitochondrial replicase is also apparently responsible for the relevant DNA synthetic reactions in these processes. Pol gamma comprises a catalytic core in a heterodimeric complex with an accessory subunit. The two-subunit holoenzyme is an efficient and processive polymerase, which exhibits high fidelity in nucleotide selection and incorporation while proofreading errors with its intrinsic 3' 5' exonuclease. Incorporation of nucleotide analogs followed by proofreading failure leads to mitochondrial toxicity in antiviral therapy, and misincorporation during DNA replication leads to mitochondrial mutagenesis and dysfunction. This review describes our current understanding of pol gamma biochemistry and biology, and it introduces other key proteins that function at the mitochondrial DNA replication fork.

Amino Acid Sequence↗

PubMed2007

A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

Binding Sites↗

PubMed1983

Purified DNA polymerase gamma replicates bovine parvovirus DNA to a unit-length product.

DNA polymerase gamma, purified from fetal bovine liver, replicated virion single-stranded DNA from bovine parvovirus to a unit-length double-stranded DNA molecule. This product was not nicked and was covalently linked to the 3' hairpin primer. The reaction was inhibited by dideoxythymidine 5'-triphosphate, but was unaffected by ATP or aphidicolin. Double-stranded viral DNA was not a functional template for purified DNA polymerase gamma.

Adenosine Triphosphate↗