Intracellular concentrations of the four deoxyribonucleoside triphosphates (dNTPs) are closely regulated, and imbalances in the four dNTP pools have genotoxic consequences. Replication errors leading to mutations can occur, for example, if one dNTP in excess drives formation of a non-Watson-Crick base pair or if it forces replicative DNA chain elongation past a mismatch before DNA polymerase can correct the error by 3' exonuclease proofreading. This review focuses on developments since 1994, when the field was last reviewed comprehensively. Emphasis is placed on the following topics: 1) novel aspects of dNTP pool regulation, 2) dNTP pool asymmetries as mutagenic determinants, 3) dNTP metabolism and hypermutagenesis of retroviral genomes, 4) dNTP metabolism and mutagenesis in the mitochondrial genome, 5) chemical modification of nucleotides as a premutagenic event, 6) relationships between dNTP metabolism, genome stability, aging, and cancer.

To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

Cancer was recognized as a genetic disease at least four decades ago, with the realization that the spontaneous mutation rate must increase early in tumorigenesis to account for the many mutations in tumour cells compared with their progenitor pre-malignant cells. Abnormalities in the deoxyribonucleotide pool have long been recognized as determinants of DNA replication fidelity, and hence may contribute to mutagenic processes that are involved in carcinogenesis. In addition, many anticancer agents antagonize deoxyribonucleotide metabolism. Here, we consider the extent to which aspects of deoxyribonucleotide metabolism contribute to our understanding of both carcinogenesis and to the effective use of anticancer agents.

The relationship between dNTP levels and DNA synthesis was investigated using ␣ factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G 1 /S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G 1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37°C before being released to 25°C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G 1 levels. Accumulation of dNTPs at G 1 /S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25°C were able to replicate when subsequently switched to 37°C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold. Hydroxyurea (HU)1 is a potent inhibitor of the enzyme ribonucleotide reductase (RNR) and inhibits DNA replication in a wide variety of cells, including Saccharomyces cerevisiae (1). The simplest explanation for HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. HU treatment has been shown to reduce the purine dNTP pools in a variety of mammalian cells (2-7); however, conflicting data exist concerning its modulation of pyrimidine dNTP pool levels. Furthermore, even for purine dNTPs, HU has only rarely been shown to cause a complete depletion of the dGTP or dATP pools (2-4). More commonly, HU results in only partial depletion of the purine dNTP pools (5-7). The complicated, often reciprocal, changes in individual dNTP pools that occur in HU-treated mammalian cells may be due to the compensatory activities of deoxyribonucleotide salvage pathways in higher eukaryotes. Budding yeast offers a simpler system in which to study the mechanism by which HU affects replication. Yeast possess no deoxyribonucleoside kinase activities, and thus deoxyribonucleotide synthesis is entirely dependent on ribonucleotide reductase. Also, yeast can easily be synchronized in G 1 using mating pheromone, and the availability of several temperature-sensitive cdc mutations allows cell cycle progression to be reversibly halted at specific points throughout the cell cycle (8). Reciprocal switch experiments in yeast have ordered the execution point of several cdc genes with respect to the HU-sensitive step during the cell cycle (9). Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the gene...

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.

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...

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SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to ␣-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated ␣-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo-and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein.The mitochondria of Slc25a19 ؊/؊ and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the ␣-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.development ͉ mitochondrial transporter ͉ mouse model ͉ thiamine pyrophosphate deficiency ͉ neural tube defect