Deoxyribonucleotide salvage falls short in whole animals.

Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells and with purified proteins. In this study, Tran et al. use Cre-Lox technology to inactivate RNR large subunit expression in heart and skeletal muscle of mouse embryos. Analysis of these mutants paints a picture of dNTP regulation in whole animals quite different from that seen in studies of purified proteins and cultured cells.

Deoxyribonucleotides as genetic and metabolic regulators.

For >35 yr, we have known that the accuracy of DNA replication is controlled in large part by the relative concentrations of the 4 canonical deoxyribonucleoside 5'-triphosphates (dNTPs) at the replisome. Since this field was last reviewed, ∼8 yr ago, there has been increased understanding of the mutagenic pathways as they occur in living cells. At the same time, aspects of deoxyribonucleotide metabolism have been shown to be critically involved in processes as diverse as cell cycle control, protooncogene expression, cellular defense against HIV infection, replication rate control, telomere length control, and mitochondrial function. Evidence supports a relationship between dNTP pools and microsatellite repeat instability. Relationships between dNTP synthesis and breakdown in controlling steady-state pools have become better defined. In addition, new experimental approaches have allowed definitive analysis of mutational pathways induced by dNTP pool abnormalities, both in Escherichia coli and in yeast. Finally, ribonucleoside triphosphate (rNTP) pools have been shown to be critical determinants of DNA replication fidelity. These developments are discussed in this review article.

Initiation of genome instability and preneoplastic processes through loss of Fhit expression.

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.

Ribonucleotide reductase association with mammalian liver mitochondria.

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes to the elevated mutation rate for the mitochondrial genome as compared with the nuclear genome. To understand this asymmetry, we must identify pathways for synthesis and accumulation of dNTPs within mitochondria. We have identified ribonucleotide reductase activity specifically associated with mammalian tissue mitochondria. Examination of immunoprecipitated proteins by mass spectrometry revealed R1, the large ribonucleotide reductase subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang and Bogenhagen (Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791-25802). Moreover, incubation of respiring rat liver mitochondria with [(14)C]cytidine diphosphate leads to accumulation of radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria. Comparable results were seen with [(14)C]guanosine diphosphate. Ribonucleotide reduction within the mitochondrion, as well as outside the organelle, needs to be considered as a possibly significant contributor to mitochondrial dNTP pools.

Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence.

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS.

Effects of a mitochondrial mutator mutation in yeast POS5 NADH kinase on mitochondrial nucleotides.

Saccharomyces cerevisiae contains three NADH/NAD(+) kinases, one of which is localized in mitochondria and phosphorylates NADH in preference to NAD(+). Strand et al. reported that a yeast mutation in POS5, which encodes the mitochondrial NADH kinase, is a mutator, specific for mitochondrial genes (Strand, M. K., Stuart, G. R., Longley, M. J., Graziewicz, M. A., Dominick, O. C., and Copeland, W. C. (2003) Eukaryot. Cell 2, 809-820). Because of the involvement of NADPH in deoxyribonucleotide biosynthesis, we asked whether mitochondria in a pos5 deletion mutant contain abnormal deoxyribonucleoside triphosphate (dNTP) pools. We found the pools of the four dNTPs to be more than doubled in mutant mitochondrial extracts relative to wild-type mitochondrial extracts. This might partly explain the mitochondrial mutator phenotype. However, the loss of antioxidant protection is also likely to be significant. To this end, we measured pyridine nucleotide pools in mutant and wild-type mitochondrial extracts and found NADPH levels to be diminished by ∼4-fold in Δpos5 mitochondrial extracts, with NADP(+) diminished to a lesser degree. Our data suggest that both dNTP abnormalities and lack of antioxidant protection contribute to elevated mitochondrial gene mutagenesis in cells lacking the mitochondrial NADH kinase. The data also confirm previous reports of the specific function of Pos5p in mitochondrial NADP(+) and NADPH biosynthesis.

Nucleoside triphosphate pool asymmetry in mammalian mitochondria.

Our laboratory has reported that deoxyribonucleoside triphosphate (dNTP) pools in rat tissue mitochondria are highly asymmetric, with dGTP predominating, and that the imbalance probably contributes toward the high spontaneous mutation rate of the mitochondrial genome. Ferraro et al. (Ferraro, P., Nicolosi, L., Bernardi, P., Reichard, P., and Bianchi, V. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 18586-18591) have challenged these findings, based upon their studies of mouse liver mitochondria. Moreover, they have identified a potential artifact in the DNA polymerase-based assay for dNTPs, based upon overestimation of dGTP when GTP levels in extracts are much higher than dGTP levels. We measured ribonucleoside triphosphate (rNTP) pools in rat mitochondrial extracts and found that GTP pools exceed dGTP pools by 50-fold or less, not enough to interfere with the dGTP assay. Analysis of dNTP pools in state 3 mitochondria, after incubation with ADP and oxidizable substrates, gave similar results. We confirmed our earlier finding that rat mitochondrial dNTP pools are highly asymmetric. dNTP pools in cytosolic extracts are uniformly low, suggesting that the dNTP pool asymmetry arises within the mitochondrion. Moreover, we found rat tissue rNTP pools to be even more highly asymmetric, with ATP, for example, at least 2 orders of magnitude more abundant than CTP in liver extracts. This finding raises the possibility that transcription of the mitochondrial genome is more error-prone than transcription in the nucleus.

Reactive oxygen species-independent oxidation of thioredoxin in hypoxia: inactivation of ribonucleotide reductase and redox-mediated checkpoint control.

We have investigated the role of cellular redox state on the regulation of cell cycle in hypoxia and shown that whereas cells expressing mutant thioredoxin (Trx) or a normal level of Trx undergo increased apoptosis, cells overexpressing Trx are protected against apoptosis. We show that hypoxia activates p53 and Chk1/Chk2 proteins in cells expressing normal or mutant Trx but not in cells overexpressing Trx. We also show that the activity of ribonucleotide reductase decreases in hypoxia in cells expressing redox-inactive Trx. Although hypoxia has been shown to induce reactive oxygen species (ROS) generation in the mitochondria resulting in enhanced p53 expression, our data demonstrate that hypoxia-induced p53 expression and phosphorylation are independent of ROS. Furthermore, hypoxia induces oxidation of Trx, and this oxidation is potentiated in the presence of 6-aminonicotinamide, an inhibitor of glucose-6-phosphate dehydrogenase. Taken together our study shows that Trx redox state is modulated in hypoxia independent of ROS and is a critical determinant of cell cycle regulation.

p53 mediates senescence-like arrest induced by chronic replicational stress.

Previous studies have shown that exposure of cells to high levels of replicational stress leads to permanent proliferation arrest that does not require p53. We have examined cellular responses to therapeutically relevant low levels of replicational stress that allow limited proliferation. Chronic exposure to low concentrations of hydroxyurea, aphidicolin, or etoposide induced irreversible cell cycle arrest after several population doublings. Inhibition of p53 activity antagonized this arrest and enhanced the long-term proliferation of p53 mutant cells. p21CIP1 was found to be a critical p53 target for arrest induced by hydroxyurea or aphidicolin, but not etoposide, as judged by the ability of p21CIP1 suppression to mimic the effects of p53 disruption. Suppression of Rad51 expression, required for homologous recombination repair, blocked the ability of mutant p53 to antagonize arrest induced by etoposide, but not aphidicolin. Thus, the ability of mutant p53 to prevent arrest induced by replicational stress per se is primarily dependent on preventing p21CIP1 up-regulation. However, when replication stress is associated with DNA strand breaks (such as with etoposide), up-regulation of homologous recombination repair in response to p53 disruption becomes important. Since replicational stress leads to clonal selection of cells with p53 mutations, our results highlight the potential importance of chronic replicational stress in promoting cancer development.

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity.

Replication of the mitochondrial genome by DNA polymerase gamma requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from approximately 1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase gamma. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3' exonuclease activity of pol gamma. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.

E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin.

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.

Measuring DNA precursor pools in mitochondria.

The ability to measure molar concentrations of deoxyribonucleoside 5'-triphosphates (dNTPs) within the mitochondrial matrix is important for several reasons. First, the spontaneous mutation rate for the mitochondrial genome is much higher than that for the nuclear genome, and dNTP concentrations are known determinants of DNA replication fidelity. Second, several human mitochondrial diseases involve perturbations of nucleotide metabolism, and dNTP pool analysis can help us to understand the consequences of these abnormalities. Third, it is important to understand how mtDNA is supplied with precursors in non-cycling cells, where the cytosolic machinery that supplies dNTPs for nuclear replication is downregulated. Fourth, the toxicity of several antiviral nucleoside analogs involves their metabolic activation within mitochondria, and dNTP pool analyses can help us to understand the processes leading to toxicity. Analyses of dNTP pools in whole-cell extracts from tissues or cultured cells are carried out either by HPLC or by an enzymatic method using DNA polymerase and defined templates. Because dNTP pools are much smaller in mitochondria than in whole cells, HPLC lacks the sensitivity needed for these measurements. The enzymatic method possesses sufficient sensitivity and is the method described in this chapter.

Maintaining precursor pools for mitochondrial DNA replication.

Among the human diseases that result from abnormalities in mitochondrial genome stability or maintenance are several that result from mutations affecting enzymes of deoxyribonucleoside triphosphate (dNTP) metabolism. In addition, it is evident that the toxicity of antiviral nucleoside analogs is determined in part by the extent to which their intracellular conversion to dNTP analogs occurs within the mitochondrion. Finally, recent work from this laboratory has shown considerable variation among different mammalian tissues with respect to mitochondrial dNTP pool sizes and has suggested that natural asymmetries in mitochondrial dNTP concentrations may contribute to the high rates at which the mitochondrial genome undergoes mutation. These factors suggest that much more information is needed about maintenance and regulation of dNTP pools within mammalian mitochondria. This review summarizes our current understanding and suggests directions for future research.

DNA precursor metabolism and genomic stability.

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.

The news of science, a colloquium-style course designed to promote lifelong scientific awareness.

It is generally agreed that informed citizens in a republic such as the United States should maintain broad awareness of current developments in science and technology. This paper describes a colloquium-style course, The News of Science, designed to stimulate in undergraduate students a desire for such awareness, and to present a convenient means for doing so. The course, which has been offered since 2000 at Oregon State University, requires students to read Science magazine and to present oral reports on articles of their choosing. Each student in the course is required to read all of the articles selected for oral presentation, and to contribute toward discussion of each talk.

Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli.

Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.

The Most Interesting Enzyme in the World.

Ribonucleotide reductases of the class I family are α2ß2 tetramers. Like all RNRs they are subject to allosteric control mechanisms affecting activity and specificity. In this issue of Structure, Johansson et al. (2016) present a structural analysis of an unusual mode of activity site regulation.