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J. Biol. Chem., Vol. 277, Issue 18, 15225-15228, May 3, 2002

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ACCELERATED PUBLICATION
Active Site Mutation in DNA Polymerase  Associated with Progressive External Ophthalmoplegia Causes Error-prone DNA Synthesis^*

Mikhail V. Ponamarev, Matthew J. Longley, Dinh Nguyen, Thomas A. Kunkel^§, and William C. Copeland^¶

From the  Laboratory of Molecular Genetics and ^§ Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, February 15, 2002, and in revised form, March 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mtDNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase  (pol ). Since Tyr-955 is a highly conserved residue critical for nucleotide recognition among family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (k_cat) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (K_m). The Y955C derivative is 2-fold less accurate for base pair substitutions than wild-type pol  despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10-100-fold. The error-prone DNA synthesis observed for the Y955C pol  is consistent with the accumulation of mtDNA mutations in patients with PEO.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Disruption of mitochondrial energy metabolism causes mitochondrial disorders that play a central role in many degenerative diseases, aging, and cancer. Hundreds of mitochondrial and nuclear gene products are required for the proper functioning of the mitochondria. Accordingly heritable mitochondrial diseases exhibit both maternal and Mendelian modes of inheritance with considerable genetic heterogeneity (1-3).

Progressive external ophthalmoplegia (PEO)¹ and mitochondrial neurogastrointestinal encephalomyopathy belong to a subclass of autosomal mitochondrial disorders associated with depletion of the mitochondrial genome and/or the accumulation of mutations and deletions within mtDNA (1, 4-6). Within the last two years, several nuclear genes controlling maintenance of mtDNA have been identified at disease loci, including the genes for adenine nucleotide translocator 1 (ANT1) at locus 4q34-35 (7), thymidine phosphorylase at locus 22q13.32-qter (8), a putative mitochondrial helicase (Twinkle) at locus 10q24 (9), an unidentified gene at locus 3p14-21 (10), and the sole mitochondrial DNA polymerase (pol ) at locus 15q22-26 (11). Sequence analysis through the pol  gene (12) in a Belgian pedigree with dominant PEO identified a heterozygous A to G mutation at codon 955 (Y955C) (11).

Located in the active site of pol , Tyr-955 is a highly conserved residue among a wide variety of DNA polymerases. As a family A DNA polymerase, pol  is related to Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase, and amino acid sequence alignments reveal that Tyr-955 in pol  is equivalent to Tyr-766 in E. coli pol I and Tyr-530 in T7 DNA polymerase (see Fig. 1A). The three-dimensional structure of T7 DNA polymerase (13) in a ternary complex with DNA and a nucleoside triphosphate places this conserved tyrosine residue in close proximity to the incoming dNTP (see Fig. 1B). Functionally Tyr-530 in T7 DNA polymerase hydrogen bonds with Glu-480 to form part of the binding pocket for the incoming dNTP and to help discriminate against ribonucleotides (13). Substitution of Tyr-766 in E. coli pol I with serine has only a minor effect on K_m(dNTP), and a slight decrease in k_cat is attributed to a 2.5-fold increase in K_d(DNA) (14). The fidelity of a Y766F substitution in the Klenow fragment does not show an appreciable increase in nucleotide misinsertion; however, substitution with alanine or serine generates an error-prone DNA polymerase attributable to decreased stringency for selection of dNTPs (15, 16). Interestingly the Y766A- and Y766S-substituted enzymes exhibited a 17-fold increase in deletions between direct repeat sequences (16). These results predict that the Y955C substitution may lower the catalytic efficiency and/or the fidelity of human pol .

We previously cloned and expressed the cDNAs for the catalytic subunit and the accessory subunit of human pol  (12, 17, 18). We have also produced a 3' to 5' exonuclease-deficient derivative of pol  to study the fidelity of DNA replication and selection of antiviral nucleotide analogs by pol  (17, 19, 20). In this report we investigate the effects of the Y955C mutation in human pol  on the kinetics and fidelity of DNA replication.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Enzyme Production-- The His₆ affinity-tagged recombinant catalytic subunit of human DNA polymerase  (p140) and the exonuclease-deficient form (p140 Exo) were purified to homogeneity as described previously (17). The His₆-tagged accessory subunit (p55) was purified to homogeneity, and the two subunit forms of the polymerase (p140·p55 and p140 Exo·p55) were reconstituted as described previously (18).

The Y955C p140 derivative was made with the QuikChange site- directed mutagenesis kit (Stratagene). The mutagenic primers 5'-CTG CCC AGC ACC ACA GAT GCG GCC GTA and 5'-TAC GGC CGC ATC TGT GGT GCT GGG CAG were used to generate Y955C in wild-type and exonuclease-deficient backgrounds in the baculovirus transfer vectors pHupVL (17) and ExopQVSL11.4 (20), respectively. The Y955C mutation was verified by complete sequencing of the pol  insert in each vector. Recombinant baculoviruses expressing the exonuclease-proficient or -deficient forms of Y955C pol  were selected and amplified, and protein was purified as described previously (17).

Polymerase Assays-- Reverse transcriptase activities of the wild-type and Y955C mutant pol  with the accessory subunit were determined at optimal salt concentration with poly(rA)·oligo(dT)_12-18 as substrate (18). Plots of rate and dTTP concentration in enzyme-limiting reactions utilizing poly(rA)·oligo(dT)_12-18 were fit with the Michaelis-Menten equation to obtain apparent kinetic constants.

Fidelity Assays-- The accuracy of DNA synthesis by human pol  was measured by copying a single-stranded region of bacteriophage M13 DNA encoding the -peptide region of the -galactosidase gene. Replication errors were scored by transfection and plating on chromogenic indicator plates to score plaque colors. The necessary bacterial strains, bacteriophage M13mp2 derivatives, and all procedures related to fidelity assays have been described elsewhere (19, 21). Gap-filling reaction mixtures (30 µl) contained 25 mM HEPES·KOH (pH 7.6), 2 mM dithiothreitol, 1 mM each of dATP, dCTP, dGTP, and dTTP, 4 mM MgCl₂, 50 µg/ml acetylated bovine serum albumin, ~150 ng of gapped M13mp2 DNA, 0.1 M NaCl, and either 20 ng of p140, 20 ng of p140 Exo, 200 ng of p140/Y955C, or 830 ng of p140/Y955C/Exo, each reconstituted with 1.3-fold molar excess of p55 accessory subunit. Reaction products were isolated by phenol extraction and ethanol precipitation, and closure of the gaps was confirmed by agarose gel electrophoresis prior to transfection. Mutation frequencies were calculated as described, and specific nucleotide changes in mutant DNAs were determined by DNA sequencing (19).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Amino acid sequence alignments (Fig. 1A) and the active site structure of T7 DNA polymerase (Fig. 1B) suggest that Tyr-955 in human pol  is crucial for dNTP binding and fidelity of nucleotide selection. To study the biochemical properties of the Y955C variant responsible for PEO, we made the Y955C substitution by site-directed mutagenesis and overproduced the mutant protein in baculovirus-infected Sf9 cells. As compared with the wild-type enzyme in the presence of the accessory subunit, the Y955C pol  exhibited a 4-fold reduction in overall DNA polymerase activity in our standard DNA polymerase assay (data not shown). Steady state kinetic analyses (Table I) indicated that the basis for the reduced activity was a 45-fold increase in the apparent K_m for the incoming dNTP from 2.1 µM (WT Exo) to 92 µM (Y955C Exo) with no reduction in k_cat and only a slight alteration in K_d(DNA). Thus, the Y955C polymerase has near wild type catalytic efficiency at higher dNTP concentrations. Since early estimates (22) suggest nucleotide triphosphate pools in the mitochondrial matrix are high enough to support competent replication by this mutant polymerase, the reduced dNTP binding affinity alone may be inadequate to explain the phenotype in clinically affected heterozygotes (wild type/Y955C). Therefore, we tested whether the Y955C mutation was affecting the fidelity of DNA replication by pol .

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Sequence alignment and nucleotide binding pocket of family A DNA polymerases. A, amino acid sequence alignment of motif B in human pol  (Hu pol), E. coli pol I (Ec polI), and T7 DNA polymerase (T7 pol). The position of the conserved Tyr residues is indicated. B, the nucleotide binding pocket of family A DNA polymerases as derived from the structure of the T7 DNA polymerase ternary complex (13). The relative positions of the primer, DNA template, the incoming ddGTP, and residue Tyr-530 (T7 DNA polymerase) or the analogous Tyr-955 (human pol ) are indicated.

View this table: [in this window] [in a new window]   Table I Effects of Y955C mutation on kinetics of DNA synthesis by pol   Apparent steady state kinetic values were determined as described under "Experimental Procedures." All values are the averages of at least two independent determinations.

We previously determined the fidelity of wild-type and exonuclease-deficient pol  with and without the p55 accessory subunit (19). Pol  is highly accurate due to high nucleotide selectivity and exonucleolytic proofreading. Indeed genetic inactivation of the exonuclease activity was needed to reveal the complete error spectrum of the polymerase function. Also the p55 accessory subunit mildly decreased fidelity by promoting extension of misinserted nucleotides, and this negative effect may be balanced by the functional benefit of enhanced processivity conferred by the p55 subunit (18, 19). The accuracy of DNA synthesis by the human Y955C pol  was measured in vitro by copying a single-stranded region of bacteriophage M13mp2 DNA encoding the -peptide of the -galactosidase gene as described previously (21). The first experiment utilized a sensitive reversion assay that measures eight different single base substitutions at a TGA codon within lacZ. In the absence of proofreading, the Y955C mutation raised the mutation frequency 42-fold from 13 × 10⁵ (WT pol ) to 550 × 10⁵ (Y955C pol ). In the exonuclease-proficient background the mutator effect of the Y955C mutation was 2-fold (0.33 × 10⁵ for WT pol  and 0.72 × 10⁵ for Y955C pol ), implying that the majority of misinsertions produced by the Y955C polymerase are proofread (Table II). Nevertheless this 2-fold increase in base substitution errors should significantly increase the mutant fraction of the heteroplasmic mtDNA pool during the preclinical onset period leading to PEO.

View this table: [in this window] [in a new window]   Table II Error frequencies of wild-type and Y955C mutant pol   Error frequencies were determined as described under "Experimental Procedures." The relative effects of the Y955C mutation are indicated in parentheses.

To gauge the broader effects of the Y955C mutation on fidelity of replication, a substrate bearing a 407-nucleotide mutational target was utilized to score all 12 possible single base substitutions, each in a variety of sequence contexts, as well as additions and deletions of 199 different template nucleotides (21). The frequencies of lacZ mutant plaques generated with and without intrinsic proofreading activity by the wild-type and Y955C forms of pol  in the presence of the 55-kDa accessory subunit are shown in Table II. Under conditions with active proofreading the Y955C mutation did not significantly change the mutation frequency, indicating that the mutations produced by the Y955C mutant polymerase are proofread by its intrinsic 3'-5' exonuclease. In the absence of proofreading the Y955C polymerase demonstrated a 7-fold higher mutant frequency as compared with the wild-type polymerase. Sequencing the DNA from mutant plaques revealed primarily single base substitution errors with 45% of the mutant plaques containing more than one mutation. When considering all mispairs, the average base substitution error rate of the Y955C polymerase was elevated 10-fold in the exonuclease-deficient background (Fig. 2). Perhaps the most striking effect of introducing the Y955C mutation was that the rates of forming point mutations due to T·dTMP and T·dGMP mispairs increased by factors of 110 and 81, respectively (Fig. 2). In fact, transition mutations caused by misinsertion of G opposite a template T represent 60% of all of the base substitution errors observed for Y955C pol  (142 of 235 mutants). Interestingly analysis of point mutations in mtDNA from PEO patients revealed A·T  G·C transitions as the most common mutation (8 of 14 cases listed in the MitoMap data base), although the autosomal defects responsible for these cases of PEO are not known.²

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Rates and classes of DNA synthesis errors made by Y955C pol . Mutation rates for the wild-type Exo pol  (solid) are from Ref. 19. Error rates for Y955C Exo pol  (stippled) were calculated as described in Ref. 21 using the forward mutant frequency data in Table II and the results of DNA sequence analysis from 216 independent lacZ mutants. The relative increase in mutation rate due to the Y955C mutation is indicated in parentheses above each class of replication error.

Pathogenic base substitutions in mtDNA are well documented for PEO.² Our in vitro data indicate that pol  makes both +1 and 1 frameshift errors at almost 20% of the base substitution error rate, although proofreading substantially reduces the frameshift error rate. The Y955C mutation increases the frameshift error rate by 4-fold, but base substitution errors remain the primary replication error catalyzed by Y955C pol . Clinical tests based on Southern blots or PCR are designed to detect deletions in mtDNA, and multiple deletions in mtDNA have become a clinical hallmark of PEO. Large deletions in mtDNA between direct repeats, such as the 4977-base pair deletion between nucleotides 8470 and 13447, are found in both dominant and recessive forms of PEO (24, 25). We present a model in which the enhanced base substitution error rate of Y955C pol  promotes deletions between direct repeats in mtDNA (Fig. 3). This mechanism invokes a misinsertion event following correct synthesis through a direct repeat sequence. Failure of the polymerase to proofread the error or to extend the mismatch favors a slippage event between the direct repeats that creates a matched DNA terminus at a downstream template sequence (Fig. 3). Large deletions between direct repeats were observed for the analogous Y766S mutation in the Klenow fragment (16), and we propose that a common T·dTMP misinsertion by Y955C pol  (Fig. 2) initiates the 4977-bp deletion observed in some PEO patients.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Misinsertion by pol  may initiate large deletions between direct repeats as observed in PEO. H and L represent the heavy and light strands of human mitochondrial DNA, respectively. Direct repeat DNA sequences are underlined. See text for description.

Mitochondrial disorders marked by depletion, deletion, or base substitution of mtDNA are most likely caused by defects in nuclear genes that function to maintain mtDNA. Genetic predisposition to the accumulation of mtDNA mutations, as in PEO patients with the Y955C allele, is consistent with the delayed-onset and progressive course of such degenerative mitochondrial diseases. In addition to our identification of exonuclease-deficient pol  (19) and Y955C pol  as mutator DNA polymerases, Zassenhaus (26) has demonstrated that transgenic overexpression of exonuclease-deficient pol  in mice is associated with cardiomyopathy and results in accumulation of point mutations, deletions, and large deletions flanked by direct repeats. Thus, mutations that reduce the fidelity of pol  cause mitochondrial disease through mutagenic replication of mtDNA. The ability of other pol  mutations linked with disease (11) to affect the fidelity of the polymerase has not been tested. Multiple mutations within the "Twinkle" gene encoding a putative mitochondrial helicase are also causally linked to dominant PEO with mtDNA deletions (9), and a dysfunctional replicative helicase (dnaB) has been shown to enhance deletions in E. coli, possibly by stalling the replication fork (27). Mutations in the nuclear genes for ANT1 or thymidine phosphorylase also induce pathogenic mutation of mtDNA (7, 8) perhaps by unbalancing or reducing the available intramitochondrial pool of deoxynucleoside triphosphates. Nucleotide pool imbalance is known to enhance base substitution errors by pol  (28, 29). Additionally Wallace (23) observed mtDNA rearrangement and increased production of reactive oxygen species in the mitochondria of ANT1^/ knockout mice, suggesting pathogenesis results from enhanced oxidative damage to mtDNA. In summary, we believe that autosomal mitochondrial disorders exhibiting point mutations, deletions, and rearrangements have a higher mtDNA mutation rate due to enhanced damage or compromised mechanisms of mtDNA maintenance, and we predict a general mechanism in which point mutations are an early event in PEO and other mitochondrial diseases affecting the integrity of mtDNA.

	ACKNOWLEDGEMENTS

We thank Drs. Katarzyna Bebenek and Farid Kadyrov for critical evaluation of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-4792; Fax: 919-541-7613; E-mail: copelan1@niehs.nih.gov.

Published, JBC Papers in Press, March 15, 2002, DOI 10.1074/jbc.C200100200

² MitoMap data base at www.gen.emory.edu/mitomap.html.

	ABBREVIATIONS

The abbreviations used are: PEO, progressive external ophthalmoplegia; pol, DNA polymerase; ANT1, adenine nucleotide translocator 1; WT, wild type.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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