The mitochondrial DNA polymerase as a target of oxidative damage

Gra̧ziewicz, Maria A.; Day, Brian J.; Copeland, William C.

doi:10.1093/nar/gkf392

Cited by 167 publications

(109 citation statements)

References 41 publications

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“…For example, Graziewicz et al (18) showed that the human ␥-polymerase, the only polymerase present and active in mitochondria, is prone to oxidative damage. This might also be the case in our experiments.…”

Section: Discussionmentioning

confidence: 99%

“…It was observed that concentrations ranging from 50 to 150 M promote DNA damage (9,10) replicative senescence (11)(12)(13), sustained p21 levels, cell cycle arrest, transient elevation of p53 protein (14), and temporary growth arrest followed by increased resistance to subsequent oxidative stress (8,15). In contrast, H 2 O 2 doses of 200 M and higher were demonstrated to induce apoptosis (16), necrosis (16,17), protein oxidation (18), as well as lesions in both nuclear and mitochondrial genomes (9,10).…”

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confidence: 99%

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Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

Santos¹,

Hunáková²,

Chen³

et al. 2003

Journal of Biological Chemistry

192

163

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In order to understand the molecular events following oxidative stress, which lead to persistence of lesions in the mtDNA, experiments were performed on normal human fibroblast (NHF) expressing human telomerase reverse transcriptase (hTERT). The formation and repair of H 2 O 2 -induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage (ϳ4 lesions/10 kb) after exposure to 200 M H 2 O 2 , which is partially repaired during a recovery period of 6 h. At the same time, the nDNA seemed to be completely resistant to damage. Cell sorting experiments revealed persistent mtDNA damage at 24 h only in the fraction of cells with low mitochondrial membrane potential (⌬⌿m). Further analysis also showed increased production of H 2 O 2 by these cells, which subsequently undergo apoptosis. This work supports a hypothesis for a feed-forward cascade of reactive oxygen species generation and mtDNA damage and also suggested a possible mechanism for persistence of lesions in the mtDNA involving a drop in ⌬⌿m, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

Santos¹,

Hunáková²,

Chen³

et al. 2003

Journal of Biological Chemistry

192

163

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“…18 In functional studies, exposure to hydrogen peroxide, a reactive oxygen species, resulted in damage to the DNA replication enzyme (polymerase g) in the mitochondria and a reduction in mtDNA copy number. 19,20 Exposure to TNF-a, an inflammatory cytokine, also generated reactive oxygen species and reduced mtDNA copy number in myocytes. 21 Cross-sectional studies of patients with CKD have shown that lower mitochondrial function, indicated by metabolites from urine and gene expression from peripheral blood, correlated with more severe CKD.…”

Section: Main Findingsmentioning

confidence: 99%

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Tin

Grams²,

Ashar

et al. 2016

JASN

118

115

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Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P,0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P,0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD.

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“…Nuclear genes include those encoding the adenine nucleotide translocator 1 (2), thymidine phosphorylase (3), mitochondrial DNA helicase (Twinkle) (4), and the catalytic subunit of mitochondrial DNA polymerase (pol 1 ␥) (5). Pol ␥ has also been shown to be a target of oxidative damage, which reduces DNA polymerase activity in the mitochondrial matrix (6).…”

mentioning

confidence: 99%

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

Luo

Kaguni

2005

Journal of Biological Chemistry

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The catalytic subunit (␣) of mitochondrial DNA polymerase (pol ␥) 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 ␥-␣. Four ␥-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 ␥. 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 singlestranded DNA-binding protein. Based on these results and crystallographic evidence showing that the templateprimer 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 ␥ may position the substrate with respect to the enzyme catalytic domains.The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for energy production. Disruption of mitochondrial energy metabolism can occur by genetic or biochemical mechanisms and is associated with human disorders including degenerative diseases, cancer, and aging (1). Mutations in both the mitochondrial genome and in nuclear genes whose products have mitochondrial functions are linked to mitochondrial disease syndromes. Nuclear genes include those encoding the adenine nucleotide translocator 1 (2), thymidine phosphorylase (3), mitochondrial DNA helicase (Twinkle) (4), and the catalytic subunit of mitochondrial DNA polymerase (pol 1 ␥) (5). Pol ␥ has also been shown to be a target of oxidative damage, which reduces DNA polymerase activity in the mitochondrial matrix (6).Pol ␥ is the only DNA polymerase known to be required for mitochondrial DNA replication in animals (7). It is a member of the family A DNA polymerase group (8, 9), of which Escherichia coli pol I is the prototype (10). The crystal structures of numerous family A DNA polymerases have been solved, revealing a high degree of structural similarity among its members (11)(12)(13)(14)(15)(16)(17). The interaction between DNA polymerase and template-primer DNA has also been revealed in molecular detail by structural determination of pol:DNA co-crystals and in biochemical studies. The pol domain comprises palm, fingers, and thumb subdomains that are separated from the 3Ј-5Ј exonuclease (exo) domain by a cleft that binds the template-primer. Wher...

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The mitochondrial DNA polymerase as a target of oxidative damage

Cited by 167 publications

References 41 publications

Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

Cell Sorting Experiments Link Persistent Mitochondrial DNA Damage with Loss of Mitochondrial Membrane Potential and Apoptotic Cell Death

Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

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