Analysis of phylogenetically reconstructed mutational spectra in human mitochondrial DNA control region

Analysis of mutations in mitochondrial DNA is an important issue in population and evolutionary genetics. To study spontaneous base substitutions in human mitochondrial DNA we reconstructed the mutational spectra of the hypervariable segments I and II (HVS I and II) using published data on polymorphisms from various human populations. An excess of pyrimidine transitions was found both in HVS I and II regions. By means of classification analysis numerous mutational hotspots were revealed in these spectra. Context analysis of hotspots revealed a complex influence of neighboring bases on mutagenesis in the HVS I region. Further statistical analysis suggested that a transient misalignment dislocation mutagenesis operating in monotonous runs of nucleotides play an important role for generating base substitutions in mitochondrial DNA and define context properties of mtDNA. Our results suggest that dislocation mutagenesis in HVS I and II is a fingerprint of errors produced by DNA polymerase gamma in the course of human mitochondrial DNA replication     

Construction of transgenic mice with tissue-specific acceleration of mitochondrial DNA mutagenesis

Transgenic mice having rapid accumulation of mitochondrial DNA (mtDNA) mutations specifically in the heart were created. These mice contained a transgene encoding a proofreading-deficient, mouse mitochondrial DNA polymerase (pol gamma) driven by the promoter for the cardiac-specific alpha-myosin heavy chain. Starting shortly after birth greater than 95% of all pol gamma mRNA in the heart was transgene derived; expression in other tissues was low or absent. Mutations in cardiac mtDNA began to accumulate by 7 days after birth. At 1 month of age the frequency of point mutations was 0.014% as determined by DNA sequencing of cloned mtDNA. By long-extension PCR multiple different deletion mutations that had removed several thousand basepairs of genomic sequence were also detected. Sequencing of two deletion molecules showed that one was flanked at the breakpoint by direct repeat sequences. The expression of proofreading-deficient pol gamma had no apparent deleterious effect on mitochondrial DNA and protein content, gene expression, or respiratory function. However, associated with the rise in mtDNA mutation levels was the development of cardiomyopathy as evidenced by enlarged hearts in the transgenic mice. These mice may prove to be useful models to study the pathogenic effects of elevated levels of mitochondrial DNA mutations in specific tissues.     

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.     

Mutations in the spacer region of Drosophila mitochondrial DNA polymerase affect DNA binding, processivity, and the balance between Pol and Exo function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.     

Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma

Mitochondrial DNA (mtDNA) mutations and deletions are frequently observed in cancer, and contribute to altered energy metabolism, increased reactive oxygen species (ROS), and attenuated apoptotic response to anticancer agents. The mechanisms by which cells maintain mitochondrial genomic integrity and the reason why cancer cells exhibit more frequent mtDNA mutations remain unclear. Here, we report that the tumor suppressor molecule p53 has a novel role in maintaining mitochondrial genetic stability through its ability to translocate to mitochondria and interact with mtDNA polymerase gamma (pol gamma) in response to mtDNA damage induced by exogenous and endogenous insults including ROS. The p53 protein physically interacts with mtDNA and pol gamma, and enhances the DNA replication function of pol gamma. Loss of p53 results in a significant increase in mtDNA vulnerability to damage, leading to increased frequency of in vivo mtDNA mutations, which are reversed by stable transfection of wild-type p53. This study provides a mechanistic explanation for the accelerating genetic instability and increased ROS stress in cancer cells associated with loss of p53.     

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.     

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.     

Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis

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 gamma (pol gamma). 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 gamma 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 gamma is consistent with the accumulation of mtDNA mutations in patients with PEO.     

Molecular insights into NRTI inhibition and mitochondrial toxicity revealed from a structural model of the human mitochondrial DNA polymerase

NRTI-based therapy used to treat AIDS can cause mitochondrial toxicity resulting from the incorporation of NRTIs into mitochondrial DNA by DNA polymerase gamma (pol gamma). Pol gamma has poor discrimination against many of the currently used NRTIs resulting in aborted DNA synthesis and subsequent depletion of mtDNA. Pol gamma readily incorporates ddCTP, ddITP and D4T-TP with an efficiency similar to the incorporation of normal nucleotides, whereas AZT-TP, CBV-TP, 3TC-TP and PMPApp act as moderate inhibitors to DNA synthesis. We have sought a structural explanation for the unique selection for NRTIs by the human pol gamma. A structural model of the human pol gamma was developed to ascertain the role of active site amino acids. One residue in particular, Y951 in motif B, is primarily responsible for the selection of dideoxynucleotides and D4T-TP. Our structural model of the human pol gamma should assist in rational design of antiviral nucleoside analogs with higher specificity for HIV-RT and minimal selection and incorporation into mitochondrial DNA.     

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.     

Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.     

Direct measurement of mutational spectra in humans

By combining high fidelity in vitro DNA amplification and mutant DNA sequence separation by denaturing gradient gel electrophoresis, we are able to directly observe mutational hotspots in human genomic DNA. Our technological development has progressed through the stage of identifying mutant sequences in independently derived, 6-thioguanine-resistant human B cells. We are now analyzing uncloned, complex populations derived from several thousand 6-thioguanine-resistant cells and report preliminary data concerning the mutational spectra of benzo[a]pyrene diol epoxide and ultraviolet light in exon 3 of the hypoxanthine-guanine phosphoribosyltransferase gene. In addition, the approach appears to be general for any gene sequence for which a means to select mutants exists. The more global need to eliminate phenotypic selection is, however, our primary impetus. Our analysis leads us to conclude that no known in vitro DNA polymerase has sufficient fidelity to permit direct observation of unselected mutants. Therefore, an additional change in technology will be necessary to observe nonselected mutant DNA sequences at the low frequencies found in human tissues.     

DNA polymerase η mutational signatures are found in a variety of different types of cancer

DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.     

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.     

p53 mutational spectra and the role of methylated CpG sequences

The occurrence of tumor-specific mutational spectra in the p53 mutation database provides indirect evidence that implicates certain exogenous and possibly endogenous mutagenic events in human carcinogenesis. In some cases, the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.e. hotspots and types of mutations of certain cancers, most notably for nonmelanoma skin cancers and lung cancers in smokers. This concept has been validated by experiments with sunlight and cigarette smoke components representing the polycyclic aromatic hydrocarbon class of carcinogens. A disproportionally high number of mutations in p53 (and other genes) are found at methylated CpG dinucleotides. These sequences are particularly prone to mutagenesis involving endogenous events as well as modification by exogenous carcinogens.     

Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans

Constant denaturant electrophoresis is a DNA separation technique based on the principle of cooperative melting equilibrium. DNA sequences with distinct high and low melting domains can be utilized to separate and identify molecules differing by only one base pair in the lower melting domain. Combined with capillary gel electrophoresis and when coupled with high fidelity DNA amplification, this approach can detect mutants at a fraction of 10⁻⁶. Modifications to the capillary elecctrophoretic system have also increased DNA loading capacity which allows for analysis of rare mutations in a large, heterogeneous population such as DNA samples derived from human tissues. Employment of this technology has determined the first mutational spectrum in human cells and tissues in a mitochondrial sequence without phenotypic selection of mutants.     

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.     

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.     

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 EM structure of human DNA polymerase gamma reveals a localized contact between the catalytic and accessory subunits

We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol gammaA, and of the holoenzyme including a dimeric accessory factor, pol gammaB(2), permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L(549) of pol gammaA upon binding of the accessory subunit. This interaction region is near residue 467 of pol gammaA, where a disease-related mutation has been reported to impair binding of the B subunit. One pol gammaB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol gamma is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.