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Abstract

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase gamma (POLgammaA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLgammaA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLgammaA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLgammaA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLgammaA:Y955C enzyme will compete with wild-type POLgammaA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLgammaA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.