Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we describe a simple and rapid protocol for reliable quantitation of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the traditional polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been of limited use for the accurate determination of ratios of mutant and wild type molecules, largely owing to the formation of heteroduplex molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then digested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN analysis software. The amount of mutant mtDNA is readily estimated from fluorescence intensities of the wild-type and mutant mtDNA fragments corrected for incomplete digestion as monitored by a homologous control fragment. The accuracy of the improved protocol was determined by constructing standard curves obtained from defined mixtures of genomic DNA containing homoplasmic wild-type and mutant mtDNA. The expected values were obtained, with an observed correlation coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screening of five patients and unaffected subjects carrying the guanine to adenine transition at the nucleotide 3460 of the mitochondrial genome responsible for the mitochondrial disorder of Leber's hereditary optic neuropathy.
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PMID:Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis. 1140 78

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.
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PMID:Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis. 1189 78

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.
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PMID:Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia. 1525 72

POLG is the human gene that encodes the catalytic subunit of DNA polymerase gamma (Pol gamma), the replicase for human mitochondrial DNA (mtDNA). A POLG Y955C point mutation causes human chronic progressive external ophthalmoplegia (CPEO), a mitochondrial disease with eye muscle weakness and mtDNA defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.
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PMID:Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma. 1748 95

We report a patient with an autosomal dominant chronic progressive external ophthalmoplegia phenotype associated with multiple mtDNA deletions in muscle from a family in which linkage analysis excluded mutations in DNA polymerase gamma (POLG), adenine nucleotide translocase (ANT-1) or C10orf2 (Twinkle). She presented with prominent Parkinsonism characterized by prolonged benefit from levodopa (L-dopa) and the later development of L-dopa induced dyskinesias and motor fluctuations. Thus L-dopa responsiveness, L-dopa induced dyskinesias and motor fluctuations may also occur in atypical Parkinsonism of mitochondrial disease, just as they may in multiple system atrophy.
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PMID:Levodopa response in Parkinsonism with multiple mitochondrial DNA deletions. 1735 42

Leigh syndrome is a common clinical manifestation in children with mitochondrial disease and other types of inborn errors of metabolism. We characterised clinical symptoms, prognosis, respiratory chain function and performed extensive genetic analysis of 25 Swedish children suffering from Leigh syndrome with the aim to obtain insights into the molecular pathophysiology and to provide a rationale for genetic counselling. We reviewed the clinical history of all patients and used muscle biopsies in order to perform molecular, biochemical and genetic investigations, including sequencing the entire mitochondrial DNA (mtDNA), the mitochondrial DNA polymerase (POLGA) gene and the surfeit locus protein 1 (SURF1) gene. Respiratory chain enzyme activity measurements identified five patients with isolated complex I deficiency and five with combined enzyme deficiencies. No patient presented with isolated complex IV deficiency. Seven patients had a decreased ATP production rate. Extensive sequence analysis identified eight patients with pathogenic mtDNA mutations and one patient with mutations in POLGA. Mutations of mtDNA are a common cause of LS and mtDNA analysis should always be included in the diagnosis of LS patients, whereas SURF1 mutations are not a common cause of LS in Sweden. Unexpectedly, age of onset, clinical symptoms and prognosis did not reveal any clear differences in LS patients with mtDNA or nuclear DNA mutations.
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PMID:MtDNA mutations are a common cause of severe disease phenotypes in children with Leigh syndrome. 1910 52

Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol gamma), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol gamma revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g-->a; T851A, c.2551a-->g; R852C, c.2554c-->t; R853Q, c.2558g-->a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g-->t and T885S, c.2653a-->t). Biochemical characterization of purified, recombinant forms of pol gamma revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50-70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol gamma accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol gamma and examines the consequences of mitochondrial disease mutations in this region.
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PMID:Disease mutations in the human mitochondrial DNA polymerase thumb subdomain impart severe defects in mitochondrial DNA replication. 1947 85

In this issue, Lee et al. (2009) present a crystal structure of the human mitochondrial DNA polymerase (POLgamma). The structure of this heterotrimeric enzyme lays a foundation for understanding how POLgamma mutations cause human mitochondrial disease and why some antiviral nucleoside analogs cause cellular toxicity.
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PMID:Structure casts light on mtDNA replication. 1983 34

Since mutations in mitochondrial DNA (mtDNA) have been shown to be a cause of many mitochondrial diseases as well as aging, it is important to understand the origin of these mutations and how replication proteins modulate this process. DNA polymerase gamma (pol gamma) is the polymerase that is responsible for replication and repair of mtDNA. Pol gamma has three main roles in mtDNA maintenance and mutagenesis. As the only known DNA polymerase in mitochondria, pol gamma is required for all replication and repair functions and is the main source of errors produced in human mtDNA. Pol gamma is also sensitive to a host of antiviral nucleoside analogs used to treat HIV-1 infections, which can cause an induced mitochondrial toxicity. Finally, the gene for pol gamma, POLG, is a genetic locus for several mitochondrial disease with over 150 genetic mutations currently identified.
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PMID:The mitochondrial DNA polymerase in health and disease. 2001 84

The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 (POLG). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems to defy the common perceptions of genetics. The unique features of mitochondrial physiology are in part responsible, but POLG structure and function add to the conundrum of how one gene product can demonstrate autosomal recessive and autosomal dominant transmission, while also being responsible for pharmacogenetic disease, and exhibiting strong gene-environment interactions. The wide spectrum of clinical manifestations of POLG disease can arise from infancy to old age. The modulation of clinical findings relate in part to the molecular architecture of the POLG protein. POLG has three distinct molecular domains: exonuclease, linker, and polymerase domains. Most of the mutations leading to dominant forms of POLG disease are located in the Polymerase domain. Mutations leading to recessive inheritance are distributed in all three domains of the gene. Environmental factors like valproic acid and infection can unmask POLG disease, causing it to occur earlier in life than when not exposed to these factors. Other drugs like nucleoside reverse transcriptase inhibitors can produce genotype-specific POLG pharmacogenetic disease. Our current state of POLG understanding cannot account for many features of POLG disease. There is no answer for why the same mutation can give rise to varying diseases, disease severity, and age of onset. We introduce the term Ecogenetics in the context these features of POLG disease, to emphasize the important interactions between genes and environment in determining the expression of mitochondrial disease. In this article, we identify some of the key features that will help the reader understand POLG pathophysiology. When possible, we also identify genotype-phenotype relationships, give clues for diagnosis, and summarize the major clinical phenotypes in the spectrum of POLG disease presenting from birth to old age.
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PMID:Polymerase gamma disease through the ages. 2081 31


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