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Free keywords:
Animals
DNA, Mitochondrial/*genetics
DNA-Directed DNA Polymerase/genetics/*metabolism
Drosophila/*genetics
Exodeoxyribonucleases/genetics/*metabolism
*Genetic Engineering
Mutagenesis, Site-Directed
Mutation
Protein Subunits
Abstract:
Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLgammaA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLgammaA, and introduce alleles expressing exonuclease- (exo(-)) and polymerase-deficient (pol(-)) POLgammaA versions. The exo(-) mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol(-) mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLgammaA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLgammaA mutation disease.
