DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity

Abstract

The mutation rate of the mammalian mitochondrial genome is higher than that of the nuclear genome. Because mitochondrial and nuclear deoxyribonucleoside triphosphate (dNTP) pools are physically distinct and because dNTP concentrations influence replication fidelity, we asked whether mitochondrial dNTP pools are asymmetric with respect to each other. We report here that the concentrations of the four dNTPs are not equal in mitochondria isolated from several tissues of both young and old rats. In particular, in most tissues examined, mitochondrial dGTP concentrations are high relative to the other dNTPs. Moreover, in the presence of the biased dNTP concentrations measured in heart and skeletal muscle, the fidelity of DNA synthesis in vitro by normally highly accurate mtDNA polymerase ␥ is reduced, with error frequencies increased by as much as 3-fold, due to increased formation of template T⅐dGTP mismatches that are inefficiently corrected by proofreading. These data, plus some published data on specific mitochondrial mutations seen in human diseases, are consistent with the hypothesis that normal intramitochondrial dNTP pool asymmetries may contribute to spontaneous mutagenesis in the mammalian mitochondrial genome. Mutations in mitochondrial DNA (mtDNA) are associated with a variety of human diseases, including neurodegenerative disorders, cardiomyopathies, and cancer (1-3). In addition, mutations in mtDNA accumulate with age (4-6) and cause premature aging in mice (7). These factors highlight the importance of understanding how genomic stability is maintained within the mitochondrion. The spontaneous mutation rate for mtDNA has been estimated to be as much as two orders of magnitude higher than that for the nuclear genome (8). Several factors could be responsible, such as excessive DNA damage generated by reactive oxygen species within the mitochondrion and͞or the absence of protection against damage that may be afforded to nuclear genes by histones. Limited DNA repair could also be a significant factor in mtDNA mutagenesis because, although mammalian mitochondria possess a base excision repair system, other DNA repair systems have not been unequivocally demonstrated in mammalian mitochondria (9-11).Analysis of human mitochondrial mutations suggests that another source of mitochondrial mutagenesis could be normal DNA replication errors (8,12). Limiting this possibility is the fact that mtDNA polymerase (pol) ␥ can replicate DNA with very high fidelity, for example, with error rates for single-base substitutions of Յ10 Ϫ5 (13-16). This accuracy results from the high nucleotide selectivity of the pol ␥ active site, combined with the ability of the intrinsic 3Ј exonuclease activity of pol ␥ to excise nucleotide misinsertions during replication.The contribution of nucleotide selectivity and proofreading to replication fidelity depends on the concentrations of the dNTP precursors available during replication. The probability of polymerase misinsertion opposite any template base will depend partly on the ratio of the...

p53 in mitochondria enhances the accuracy of DNA synthesis

“…Normal intramitochondrial dNTP pools are highly asymmetric and may reduce by three-fold the fidelity of DNA synthesis by pol g due to increased formation of dGTP:T mispairs and inefficient correction by proofreading. 31 In H1299mit, the incorporation of dGTP opposite the template T was observed with standing-start dsDNA substrate (wherein the target template residue immediately follows the 3 0 -terminal end of the primer) ( Figure 1b, lanes 2 and 3). The 17-and 18mer products were accumulated following the production of the T:G mispair.…”

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

“…As TTP becomes limiting, mitochondrial DNA replication slows, and eventually mitochondrial DNA depletion is evident. Additionally, an imbalance in the TTP pool relative to the other dNTP pools is known to increase the mitochondrial DNA mutation rate [29]. Further evaluation of this potential mechanism will require studies correlating the depletion of TTP pools with cytotoxicity, mitochondrial toxicity, and mitochondrial DNA depletion, perhaps in hepatocyte or cardiomyocyte cell culture.…”

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

“…Abs to neither Wtm1 nor Kap122 were available. Therefore, we made centromeric (low copy number) plasmids that express C-terminally epitope-tagged Wtm1-hemagglutinin (HA) 3 and Kap122-(Myc) 9 under their respective native promoters. These two plasmids were transformed either individually or together into the WT strain.…”

“…Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4)(5)(6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7)(8)(9). Eukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to ensure proper dNTP pool sizes during the normal cell-cycle progression and in response to genotoxic stress (3,(10)(11)(12)(13)(14).…”