Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity.

The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.     

A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits

Human mitochondrial DNA (mtDNA) polymerase γ (pol γ) is the sole enzyme required to replicate and maintain the integrity of the mitochondrial genome. It comprises two subunits, a catalytic p140 subunit and a smaller p55 accessory subunit encoded by the POLG2 gene. We describe the molecular characterization of a potential dominant POLG2 mutation (p.R369G) in a patient with adPEO and multiple mtDNA deletions. Biochemical studies of the recombinant mutant p55 protein showed a reduced affinity to the pol γ p140 subunit, leading to impaired processivity of the holoenzyme complex but did not show sensitivity to N-ethylmalaimide (NEM) inhibition, inferring a novel disease mechanism.     

Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders

This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases. Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma (polgamma). We investigated the fidelity of DNA replication by polgamma with and without exonucleolytic proofreading and its p55 accessory subunit. Polgamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA. Recently, several mutations in the polymerase and exonuclease domains of the human polgamma have been shown to be associated with PEO. We are analyzing the effect of these mutations on the human polgamma enzyme. In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of polgamma. These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E.coli pol I. These PEO mutations have been generated in polgamma to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis. One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families. The Y955C mutant polgamma retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP. The Y955C derivative is also much less accurate than is wild-type polgamma, with error rates for certain mismatches elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C polgamma is consistent with the accumulation of mtDNA mutations in patients with PEO. The effects of other polgamma mutations associated with PEO are discussed.     

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.     

Detection of the G to A mitochondrial DNA mutation at position 11778 in German families with Leber's hereditary optic neuropathy

Summary Leber's hereditary optic neuropathy (LHON) is characterized by acute or subacute bilateral (usually permanent) loss of central vision, caused by neuroretinal degeneration. The maternal inheritance is explained by the mitochondrial origin of the disease. Recently, a single mitochondrial DNA (mtDNA) mutation, a G to A substitution at position 11778 that converts a highly conserved arginine to histidine, has been associated with LHON. The mutation eliminates an SfaNI restriction enzyme recognition site and thus provides a method for detection of the muation by amplification, enzyme digestion and agarose gel electropheresis of polymerase chain reaction (PCR) products. Leukocyte mtDNA from 7 German families with LHON, diagnosed by clinical criteria, was tested for the presence of the G to A mutation at bp 11778. The mtDNa mutation, detected as a loss of the SfaNI site, was seen in one family. The G to A mtDNA mutation is the only known gene alteration associated with LHON so far. It has been identified in patients of different ethnic origin and recent reports strongly support the hypothesis that it represents the most frequent cause of LHON. Identification of the mtDNA replacement mutation using PCR and restriction enzyme digestion requires only a small amount of blood and can be performed rapidly. This method is thus a useful tool in the diagnosis of LHON.     

Mutation at the polymerase active site of mouse DNA polymerase delta increases genomic instability and accelerates tumorigenesis

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.     

Oligomerization of a Peptide Derived from the Transmembrane Region of the Sodium Pump γ Subunit: Effect of the Pathological Mutation G41R

The Na,K-ATPase, or sodium pump, is a ubiquitously expressed membrane-bound enzyme that controls the transmembrane (TM) gradients of sodium and potassium ions in animal cells. The enzyme comprises two subunits, α and β, and in the kidney, is also associated with a small single-spanning membrane protein, the γ subunit. This 65 amino acid residues protein has been linked to a form of dominant renal hypomagnesaemia resulting from substitution of a highly conserved glycine residue (Gly41) to arginine residue. In order to characterize the quaternary structure of the γ subunit, and effects of the G41R mutation thereupon, we synthesized a series of peptides (wild-type and mutant) that span the γ subunit TM region. Using circular dichroism spectroscopy, we show that the 32-amino acid residue peptides are random coils in aqueous buffer but spontaneously adopt an α-helical conformation in the presence of detergent micelles (sodium dodecyl sulfate, SDS, and perfluorooctanoate, PFO). Furthermore, fluorescence resonance energy transfer experiments, combined with polyacrylamide gel electrophoresis, demonstrate that while γ-TM does not self-associate in SDS, it forms oligomers in PFO, a detergent that tolerates relatively weak associations between membrane proteins. Importantly, oligomerization of γ-TM is abrogated in a peptide that contains either the disease-causing mutation G41R, or the more conservative mutation G41L. On the other hand, a peptide that contains a Gly-to-Arg substitution on a different face of the helix, at position 35, retains its ability to oligomerize. Our results provide evidence for a link between renal hypomagnesaemia and γ subunit oligomerization.     

Mitochondrial DNA defects in Saccharomyces cerevisiae caused by functional interactions between DNA polymerase gamma mutations associated with disease in human

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.     

Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.     

Effect of double mutation on thermostability of lactate oxidase

DNA shuffling was used to make a double mutant lactate oxidase (E160G/V198I LOD) in E. coli was more thermostable than both E160G single-mutant and wild-type LODs. The half-life of this E160G/V198I LOD at 70°C was about 3 times that of E160G LOD, and was about 20 times that of wild-type. In contrast, the thermostability of the V198I single-mutant LOD made by site-directed mutagenesis was almost identical to that of wild-type. This indicates that the V198I mutation alone does not affect LOD thermostability but does affect it when combined with the E160G mutation.     

A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wildtype or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg¹⁴⁰ is crucial for the structure of an enzymatically active LCAT protein.     

The Saccharomyces cerevisiae rev6-1 mutation, which inhibits both the lesion bypass and the recombination mode of DNA damage tolerance, is an allele of POL30, encoding proliferating cell nuclear antigen

The rev6-1 allele was isolated in a screen for mutants deficient for UV-induced reversion of the frameshift mutation his4-38. Preliminary testing showed that the rev6-1 mutant was substantially deficient for UV-induced reversion of arg4-17 and ilv1-92 and markedly UV sensitive. Unlike other REV genes, which encode DNA polymerases and an associated subunit, REV6 has been found to be identical to POL30, which encodes proliferating cell nuclear antigen (PCNA), the subunit of the homotrimeric sliding clamp, in which the rev6-1 mutation produces a G178S substitution. This substitution appears to abolish all DNA damage-tolerance activities normally carried out by the RAD6/RAD18 pathway, including translesion replication by DNA polymerase zeta/Rev1 and DNA polymerase eta, and the error-free, recombination-dependent component of this pathway, but has little effect on the growth rate, suggesting that G178S may prevent ubiquitination of lysine 164 in PCNA. We also find that rev6-1 mutation can be fully complemented by a centromere-containing, low copy-number plasmid carrying POL30, despite the presumed occurrence in the mutant of sliding clamp assemblies that contain between one and three G178S PCNA monomers as well as the fully wild-type species.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Dissection of two hallmarks of the open promoter complex by mutation in an RNA polymerase core subunit

Deletion of 10 evolutionarily conserved amino acids from the beta subunit of Escherichia coli RNA polymerase leads to a mutant enzyme that is unable to efficiently hold onto DNA. Open promoter complexes formed by the mutant enzyme are in rapid equilibrium with closed complexes and, unlike the wild-type complexes, are highly sensitive to the DNA competitor heparin (Martin, E., Sagitov, V., Burova, E., Nikiforov, V., and Goldfarb, A. (1992) J. Biol. Chem. 267, 20175-20180). Here we show that despite this instability, the mutant enzyme forms partially open complexes at temperatures as low as 0 degrees C when the wild-type complex is fully closed. Thus, the two hallmarks of the open promoter complex, the stability toward a challenge with DNA competitors and the sensitivity toward low temperature, can be uncoupled by mutation and may be independent in the wild-type complex. We use the high resolution structure of Thermus aquaticus RNA polymerase core to build a functional model of promoter complex formation that accounts for the observed defects of the E. coli RNA polymerase mutants.     

Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> polyhydroxyalkanoate synthase

The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC_Re). We have now carried out site-directed saturation mutagenesis of F420 of PhaC_Re and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC_Re enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC_Re enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.     

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.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.