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Mutations in human DNA polymerase ? confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients.

ABSTRACT: Human mitochondrial DNA polymerase ? (pol ?) is solely responsible for the replication and repair of the mitochondrial genome. Unsurprisingly, alterations in pol ? activity have been associated with mitochondrial diseases such as Alpers syndrome and progressive external ophthalmoplegia. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in pol ? activity has been difficult. In order to understand the relationship between disease severity in patients and enzymatic defects in vitro, we characterized the molecular mechanisms of four pol ? mutations, A957P, A957S, R1096C and R1096H, which have been found in patients suffering from aggressive Alpers syndrome to mild progressive external ophthalmoplegia. The A957P mutant showed the most striking deficiencies in the incorporation efficiency of a correct deoxyribonucleotide triphosphate (dNTP) relative to wild-type pol ?, with less, but still significant incorporation efficiency defects seen in R1096H and R1096C, and only a small decrease in incorporation efficiency observed for A957S. Importantly, this trend matches the disease severity observed in patients very well (approximated as A957P ? R1096C ? R1096H ? A957S, from most severe disease to least severe). Further, the A957P mutation conferred a two orders of magnitude loss of fidelity relative to wild-type pol ?, indicating that a buildup of mitochondrial genomic mutations may contribute to the death in infancy seen with these patients. We conclude that characterizing the unique molecular mechanisms of pol ? deficiency for physiologically important mutant enzymes is important for understanding mitochondrial disease and for predicting disease severity.

SUBMITTER: Sohl CD

PROVIDER: S-EPMC3578408 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Mutations in human DNA polymerase γ confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients.

Sohl Christal D CD Kasiviswanathan Rajesh R Copeland William C WC Anderson Karen S KS

Human molecular genetics 20121203 6

Human mitochondrial DNA polymerase γ (pol γ) is solely responsible for the replication and repair of the mitochondrial genome. Unsurprisingly, alterations in pol γ activity have been associated with mitochondrial diseases such as Alpers syndrome and progressive external ophthalmoplegia. Thus far, predicting the severity of mitochondrial disease based the magnitude of deficiency in pol γ activity has been difficult. In order to understand the relationship between disease severity in patients and ...[more]

PMID: 23208208

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Project description:DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both acrR and rpoA mutations reduced cross-link formation in vivo. We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links in vivo, and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.

Dataset Information

Mutations in human DNA polymerase ? confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients.

Publications

Mutations in human DNA polymerase γ confer unique mechanisms of catalytic deficiency that mirror the disease severity in mitochondrial disorder patients.

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