Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.     

Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma

Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K(m) and k(cat) values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), 2',3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3' termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.     

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.     

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.     

Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos greatly enhance the overall activity of pol gamma by increasing primer recognition and binding and stimulating the rate of initiation of DNA strands (Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) J. Biol. Chem. 274, 14779-14785). We show here that DNA-binding mutants of mtSSB are defective in stimulation of DNA synthesis by pol gamma. RNAi knock-down of mtSSB reduces expression to <5% of its normal level in Schneider cells, resulting in growth defects and in the depletion of mitochondrial DNA (mtDNA). Overexpression of mtSSB restores cell growth rate and the copy number of mtDNA, whereas overexpression of a DNA-binding and functionally impaired form of mtSSB neither rescues the cell growth defect nor the mtDNA depletion phenotype. Further development of Drosophila animal models, in which induced mtDNA depletion is manipulated by controlling exogenous expression of wild-type or mutant forms, will offer new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.     

Long patch base excision repair in mammalian mitochondrial genomes

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.     

Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).     

Unusual regulation of expression of the herpes simplex virus DNA polymerase gene.

During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.     

Insights into the molecular mechanism of mitochondrial toxicity by AIDS drugs

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol gamma), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2',3'-dideoxycytidine (ddC)) and Lamivudine (beta-d-(+)-2',3'-dideoxy-3'-thiacytidine ((-)3TC])). (-)3TC is the only analog containing an unnatural l(-) nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture (-)3TC is less toxic than its d(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and (-)3TC-TP on the polymerase and exonuclease activities of recombinant human Pol gamma. This analysis reveals that Pol gamma incorporates (-)3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3'-end of the DNA primer by the 3'-5'-exonuclease activity of Pol gamma were similar (0.01 s(-)1) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.     

The fidelity of human DNA polymerase gamma with and without exonucleolytic proofreading and the p55 accessory subunit

Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we investigated the fidelity of DNA synthesis by human pol gamma. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol gamma has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol gamma is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol gamma has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol gamma both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol gamma catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol gamma.     

Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.     

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.     

Protein sequences conserved in prokaryotic aminoacyl-tRNA synthetases are important for the activity of the processivity factor of human mitochondrial DNA polymerase

Previous studies have shown that the small subunit of Xenopus DNA polymerase gamma (pol gammaB) acts as a processivity factor to stimulate the 140 kDa catalytic subunit of human DNA polymerase gamma. A putative human pol gammaB initially identified by analysis of DNA sequence had not been shown to be functional, and appeared to be an incomplete clone. In this paper, we report the cloning of full-length human and mouse pol gammaB. Both human and mouse pol gammaB proteins were expressed in their mature forms, without their apparent mitochondrial localization signals, and shown to stimulate processivity of the recombinant catalytic subunit of human pol gammaA. Deletion analysis of human pol gammaB indicated that blocks of sequence conserved with prokaryotic class II aminoacyl-tRNA synthetases are necessary for activity and inter-action with human pol gammaA. Purification of DNA pol gamma from HeLa cells indicated that both proteins are associated in vivo.     

Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein. Template-primer DNA binding and initiation and elongation of DNA strand synthesis.

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol gamma and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in pol gamma are stimulated 15-20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimulation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol gamma in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol gamma catalytic efficiency is likely not accompanied by increased nucleotide turnover. DNase I footprinting of pol gamma.DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation studies show that productive complexes of the native pol gamma heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins.     

Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2-3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 > Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT](10) and [TC](11) microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences.     

Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors

Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.     

The Accessory Subunit of Xenopus laevis Mitochondrial DNA Polymerase γ Increases Processivity of the Catalytic Subunit of Human DNA Polymerase γ 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.