Mitochondrial DNA polymerase gamma is essential for mammalian embryogenesis

Nicole Hance 0 Mats I. Ekstrand 0 Aleksandra Trifunovic 0 0 Department of Medical Nutrition and Department of Biosciences at Novum, Karolinska Institute , Stockholm, Sweden Mitochondrial DNA (mtDNA) polymerase gamma (Polg) is a heterodimeric enzyme containing a Pol I-like catalytic core (PolgA) and an accessory subunit. Mutations in POLGA, affecting the stability of mtDNA, have been identified in several human pathologies such as progressive external ophthalmoplegia and Alpers' syndrome. Extensive literature shows mitochondrial toxicity effects nucleoside analogue reverse transcriptase inhibitors used in the treatment of HIV and chronic hepatitis B as a consequence of an inhibitory effect on Polg. We have previously shown that mice with an error-prone version of PolgA accumulate higher levels of somatic mtDNA mutations resulting in a premature aging phenotype. In the present paper, we demonstrate PolgA deficiency in mouse embryos causes an early developmental arrest between embryonic days 7.5 and 8.5 associated with severe mtDNA depletion. Heterozygous knockout mice have half the wild-type levels of PolgA transcripts and a slight reduction in mtDNA levels but develop normally. Surprisingly, amounts of PolgA transcripts in heterozygous knockout mice are increased in response to artificially elevated mtDNA copy number, revealing a possible regulatory link between mtDNA maintenance and PolgA expression. Our results show that Polg indeed is the only DNA polymerase capable of maintaining mtDNA in mammalian mitochondria. In addition, presence of Polg is absolutely essential for the organogenesis during mammalian embryonic development. - INTRODUCTION Fifteen distinct cellular DNA polymerases have been identified in mammalian cells but only four of these are devoted to DNA replication, whereas the rest are devoted to DNA repair and specialized DNA synthetic processes that contribute substantially to the maintenance of genetic integrity (1). Although most of these enzymes are involved in nuclear DNA repair and replication, DNA polymerase gamma (Polg) remains the only DNA polymerase found in mitochondria (reviewed in 2). As such, Polg is proposed to be uniquely responsible for all DNA synthetic reactions including both replication and repair of mitochondrial DNA (mtDNA) (2). Mitochondria are the sole organelles in animal cells that contain their own DNA. Individual cells have between 1000 and 10 000 copies of the mitochondrial genome (mtDNA) with the more energy consuming tissues, such as heart, having proportionally higher amounts. Replication of mammalian mtDNA is under relaxed control as there is no known mechanism to ensure that each molecule is replicated only once per cell cycle. Polg is not believed to directly regulate mtDNA levels, as overexpression of Polg in cell lines does not result in a corresponding increase in mtDNA copy number (3). The enzyme contains a Pol I-like catalytic core (PolgA), similar to the bacteriophage T7 DNA polymerase, in a heterodimeric complex with an accessory subunit (4 6). This accessory subunit substantially increases both the catalytic activity and the processivity of the enzyme (6). Interaction of the catalytic and accessory subunits is through multiple contact sites and it has been proposed that these contacts may enhance DNA binding and also increase the nucleotide binding of the holoenzyme (7,8). In addition, Polg exhibits a high fidelity in nucleotide selection and incorporation as well as high processivity during replication (9,10). High fidelity of the Polg is partly due to the 30 50 exonuclease activity of the catalytic subunit. It has been shown that the exonuclease activity of Polg contributes several hundred-fold to error avoidance in vivo (8). We recently described a knock-in mouse model that expresses an error-prone version of Polg (mtDNA mutator mice) (11). This mouse strain exhibits a dramatic decrease in exonuclease activity whereas Polg polymerase activity is preserved. Abolished exonuclease activity leads to a 3 5-fold increase in somatic mtDNA mutations that, in turn, cause a progressive respiratory chain deficiency and premature aging phenotypes (11). These results offer direct genetic evidence that the collective effect of a variety of somatic mtDNA mutations can cause aging. Polg is unique among the cellular replicative DNA polymerases as it is highly sensitive to inhibition by nucleoside analogue reverse transcriptase inhibitors (NRTIs) used in the treatment of HIV and chronic hepatitis B and C infections (12,13). Highly active antiviral therapy (HAART) that includes NRTIs has changed AIDS from a lethal illness to a chronic disease. Unfortunately, it was quickly seen that significant mitochondrial toxicity due to HAART was an important clinical entity and has since been shown that it is primarily a consequence of Polg inhibition. Pyrimidine NRTIs, such as AZT, fialuridine and others, cause cardiac dysfunction, hepatic failure, skeletal myopathy, lactic acidosis with defective mtDNA replication, mtDNA depletion and altered mitochondrial ultrastructure (reviewed in 13). Mutations in POLGA, the gene encoding the catalytic subunit of human mtDNA polymerase has been associated with a number of mitochondrial disorders that affect the stability of mtDNA (14,15). These mutations can lead to both mtDNA deletion and mtDNA depletion syndromes. Most common and best known are the autosomal dominant or autosomal recessive (ar) forms of familial progressive external ophthalmoplegia (15 17). Additional clinical presentations like ar sensory atactic neuropathy with dysarthria and ophthalmoplegia have been associated with POLGA mutations (18). Recently, different POLGA mutations causing the depletion of liver mtDNA have been reported in Alpers hepatophatic poliodystrophy (19,20). In addition, dominant POLGA mutations have shown to cause a severe multisystem disorder including Parkinsonism and premature menopause, which are not typical of mitochondrial disease (21). Finally, specific polymorphisms in the POLGA gene have been associated with male infertility (22). The involvement of Polg in a number of different human pathologies, its complex role in mtDNA maintenance and the still ongoing debate about the number of DNA polymerases in mitochondria, makes it a very interesting target for further analysis. In the present study, we investigated the role of Polg in mammalian development by creating PolgA deficient mice. We generated a transgenic mouse model lacking PolgA through homologous recombination in embryonic stem cells. Loss of Polg in mice leads to developmental arrest at around embryonic day (E) 7.5 associated with severe mtDNA depletion. Mice heterozygous for the PolgA null allele have only half the wild-type levels of PolgA transcripts. However, these heterozygous PolgA knockouts appear completely normal and have close to wild-type mtDNA copy number. Interestingly, mice with artificially elevated mtDNA levels show (...truncated)