Deoxyribonucleotides as Genetic and Metabolic Regulators
Mathews, C. K. (2014). Deoxyribonucleotides as genetic and metabolic regulators.
The FASEB Journal, 28(9), 3832-3840. doi:10.1096/fj.14-251249
10.1096/fj.14-251249
Federation of American Societies for Experimental Biology
Accepted Manuscript
http://cdss.library.oregonstate.edu/sa-termsofuse
Deoxyribonucleotides as Genetic and Metabolic Regulators
Christopher K. Mathews
Oregon State University
Department of Biochemistry and Biophysics
Corvallis, Oregon, USA
Correspondence
Department of Biochemistry and Biophysics
Oregon State University
2011 Agricultural & Life Sciences Bldg.
Corvallis, OR 97331-7305, USA
E-mail: mathewsc@onid.orst.edu
Running Title
dNTPs as Genetic and Metabolic Regulators
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Nonstandard abbreviations: dNMP, dNDP, dNTP, deoxyribonucleoside mono-, di-, and
triphosphate; rNMP, rNDP, rNTP, ribonucleoside mono-, di-, and triphosphate; RNR,
ribonucleoside diphosphate reductase; MMR, mismatch repair
2
Abstract
For more than 35 years we have known that the accuracy of DNA replication is controlled in
large part by the relative concentrations of the four canonical deoxyribonucleoside 5'triphosphates (dNTPs) at the replisome. Since this field was last reviewed, about eight years 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 E. 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.
Key words: mutagenesis, deoxyribonucleotides, nucleotide pools, oncogenes, cell cycle
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It is now more than three decades since investigators first established that dNTP
concentrations at replication sites are critical determinants of DNA replication fidelity and, hence,
of spontaneous mutation rates. In recent years we have learned that maintaining nucleotide pools
within controlled limits is relevant to additional cell processes, including cell cycle control,
protooncogene function, mitochondrial function, defense against virus infection, DNA mismatch
repair, and possibly telomere length determination and microsatellite repeat instability. I begin
this article by considering how current technology has illuminated mutagenic pathways resulting
from defined alterations in DNA precursor pools, and then move to other functions and processes.
Figure 1 summarizes principal reactions in dNTP biosynthesis, identifying major allosteric
control points.
MUTAGENIC MECHANISMS INFERRED FROM NUCLEOTIDE POOL VARIATIONS
Ribonucleotide reductase as the source of altered dNTP pools
It was perhaps inevitable that the earliest papers describing relationships between dNTP
pool sizes and genomic stability in living cells involved altered allosteric regulation of
ribonucleotide reductase, because of the function of this enzyme in synthesizing all four dNTPs
(1). RNR is also at the center of more recent investigations, in which sequence analysis of mutant
genes reveals defined mutagenic pathways in vivo resulting from specific pool imbalances (2,3).
Kumar et al (2) studied the effects of three mutations in loop 2 of yeast RNR, a conserved region
that connects the two allosteric control sites—the activity site and the specificity site—on the
RNR large subunit, Rnr1. By correlating pool size abnormalities in each mutant with the
mutation spectrum as revealed by sequence analysis in the CAN1 locus, conferring canavanine
resistance, Kumar et al were able to establish consistently plausible mechanisms for each
mutagenic event. Particularly important was their analysis of insertion and deletion mutations, in
which slipped-strand intermediates, previously postulated from in vitro studies, rationalized the
effects of specific pool imbalances on predicted misaligned pre-mutagenic intermediates.
Mutagenic effects, which were large, overrode both proofreading and mismatch repair, both of
which were active in these cells.
A different tack was taken in the E. coli system by Ahluwalia et al (3). These
investigators used an unbiased approach to generate mutant forms of RNR that confer a mutator
phenotype. Ahluwalia et al conducted random mutagenesis of a plasmid containing the nrdA and
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nrdB genes, which encode the large (R1) and small (R2) proteins, each of which consists of two
subunits. After transfection of the mutated plasmids into E. coli, a powerful selection technique
was used to replace the resident nrdAB system with the mutated genes, and a papillation assay
was devised to select for strains that showed elevated spontaneous mutagenesis. The 23 mutator
mutations characterized clustered in three parts of the RNR structure: (1) the activity site, which
responds to ATP and dATP; (2) the specificity site, which responds to dATP, dGTP, and dTTP;
and (3) unexpectedly, a small region of the small (R2) subunit, which lies close to the activity
site in the probable quaternary structure of the enzyme. All 23 mutator strains displayed dNTP
pool abnormalities, which corresponded well with the mutational pathways that were stimulated.
The mutants selected and the general approach should be of great value as investigators probe
the structural basis for allosteric regulation in this central enzyme.
In fact, these strains have already proven to be useful for probing mechanisms both of
mutagenesis and of regulation of RNR activity and specificity. Ahluwalia and Schaaper (4)
studied one set of the specificity-site mutators, which showed dNTP pool alterations of about
twofold, but enhancement of mutagenesis of 1000-fold or more—a phenomenon termed by the
authors an “error catastrophe.” Evidence indicated that even these modest pool changes led to
saturation of the mismatch repair activity. The MMR system appears particularly sensitive to
changes in the dGTP pool, and that may explain the longstanding observations that dGTP is
almost always the least abundant of the four canonical dNTPs.
Mutagenesis involving enzymes other than ribonucleotide reductase
Nucleoside diphosphate kinase. Mutations affecting nucleotide metabolic enzymes other
than RNR have also proven instructive. In E. coli mutations affecting ndk, the structural gene for
nucleoside diphosphate kinase, were shown some time ago to confer both nucleotide pool
abnormalities and a mutator phenotype (5). NDP kinase converts the eight canonical rNDPs and
dNDPs to the respective nucleoside triphosphates, so is involved in both DNA and RNA
synthesis. The ndk mutations caused both a dCTP elevation and a dATP depletion; mechanisms
for these effects are unknown. Nordman and Wright (6) argued that the dNTP pool alterations
were not responsible for the mutator phenotype; their data suggested that the ndk mutant
accumulated dUTP and that the mutator phenotype resulted from saturation of the DNA-uracil
repair pathway. However, Schaaper and Mathews (7) pointed out the importance of using MMRdeficient strains in this type of analysis. When MMR-deficient strains were used in a lacZ
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reversion assay (8), the ndk mutation predominantly stimulated A.T--->T.A transversions. In the
particular reporter system used, the next nucleotide was dCTP; hence, in the absence of MMR,
even a modest dCTP elevation could provoke mutagenesis by a next-nucleotide effect.
dCTP deaminase. In the same study a dcd mutation, affecting dCTP deaminase, provoked
transversion mutations by both G.C--->T.A and A.T--->T.A pathways (in E. coli the conversion
of deoxycytidine nucleotides to deoxyuridine nucleotides occurs at the triphosphate level). The
dcd mutation causes dCTP to accumulate. Again, as seen with the ndk mutants, sequence context
was critical. For both reversions that were stimulated, dCTP, which accumulates in dcd mutant
bacteria, was the next nucleotide.
Mutations in both ndk and dcd were used in a recent study by Gawel et al (9), who asked
whether DNA replication fidelity in E. coli is higher on the leading or lagging strand. Even
though in bacteria both strands are replicated by the same polymerase, previous studies had
suggested that fidelity is higher for lagging-strand replication—a seemingly counterintuitive
finding because of the more complex process of discontinuous DNA synthesis on the lagging
strand. For reversions stimulated by the ndk or dcd mutations used in the previous study, the
mutator effect will vary depending upon which strand undergoes the initial deoxyribonucleotide
incorporation, because the next nucleotide in the template will be different in each case. Results
of experiments in which reversion of each mutation was analyzed in both a leading- and a
lagging-strand context supported the previous finding of greater lagging-strand replication
accuracy.
dCMP deaminase. Deoxycytidylate deaminase is another enzyme that plays a critical role
in controlling dNTP pool sizes. Through its allosteric activation by dTTP and inhibition by dCTP,
the enzyme helps to keep in balance the pyrimidine dNTP pool. Early studies (1) suggested that
mammalian cell mutants deficient in dCMP deaminase displayed a mutator phenotype resulting
from dCTP accumulation and dTTP depletion. Kohalmi et al (10) showed that disruption of the
gene encoding dCMP deaminase in budding yeast, Saccharomyces cerevisiae, showed the
expected mutational specificity resulting from the pyrimidine dNTP imbalance. More recently
Sánchez et al (11) showed that deletion of the dCMP deaminase gene in fission yeast,
Schizosaccharomyces pombe, has a similar effect on dNTP pools but a more severe consequence
in terms of genome instability, including replication fork collapse and activation of multiple
DNA damage and checkpoint responses
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dGTP triphosphohydrolase. Another enzyme mentioned in this section is E. coli dGTP
triphosphohydrolase, an enzyme of still unknown function that cleaves dGTP to deoxyguanosine
and tripolyphosphate, but evidently only in enteric bacteria. Gawel et al (12) made the
serendipitous observation that insertional inactivation of dgt, the structural gene for this enzyme,
conferred a mutator phenotype, with a specificity toward G.C---> C.G transversions and A.T--->
G.C transitions. Both events could be stimulated by an increased dGTP pool, and that may well
result from a deficiency of an enzyme that degrades dGTP. However, dNTP pools were not
measured in this study; such data would be of great interest.
Polynucleotide phosphorylase. Intriguingly, Becket et al (13) reported that E. coli
mutations that inactivate polynucleotide phosphorylase (PNP) confer an antimutator effect seen
both for frameshift and substitution mutations. PNP catalyzes the phosphorolysis of RNA to
rNDPs, which, of course, are the immediate precursors to deoxyribonucleotides via RNR.
Whether the antimutagenic effect results from either dNTP pool changes or ribonucleotide
depletion is a question of great interest. As discussed later in this article, ribonucleotide
metabolism has recently been assigned a much larger role in DNA replication fidelity than
previously suspected. On the other hand, dNTP pool changes may well be involved. Using an
assay procedure that permits simultaneous determination of all four reactions catalyzed by
ribonucleotide reducatase, our laboratory showed (14) that the activities of this enzyme are
probably controlled by relative concentrations of the four rNDP substrates in vivo. Hence, a
partial depletion of rNDP pools could decrease in vivo flux rates through RNR with a consequent
depletion of dNTPs. As discussed elsewhere in this article, balanced dNTP depletions can
enhance replication accuracy, by allowing for enhanced proofreading during DNA replication. In
any event, it will be of great interest to learn the effects of polynucleotide phosphorylase
mutations upon both ribonucleotide and deoxyribonucleotide pools.
RIBONUCLEOTIDE METABOLISM AND DNA REPLICATION FIDELITY
It has long been known that DNA polymerases will accept ribonucleoside triphosphates
as substrates, albeit poorly, and that rNTP pools are much larger than dNTP pools in all cells
examined to date. However, it is only recently that investigators have asked whether
ribonucleotide incorporation into DNA in living cells might be a source of genomic instability.
Nick McElhinny et al (15) examined the situation in yeast, where they found rNTP pools to
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exceed the respective dNTP pools by factors of 36 to 190. Replicative DNA polymerases
preferred dNTPs over rNTPs by several hundred- to several thousandfold, depending upon
sequence context and the specific polymerase. The data suggested that one round of replication
in vivo could result in 10,000 rNMPs incorporated into DNA, making ribonucleotide
incorporation potentially the most abundant endogenous source of DNA damage. Data suggested
(16, 17) that genomic instability resulted from strand misalignment during replication, due to the
tendency of incorporated ribonucleotides to drive DNA from the B to the A conformation. rNTP
incorporation has been shown also to slow the rate of DNA replication, due primarily to DNA
polymerase stalling, particularly at sites where two or more consecutive ribonucleotides have
been incorporated (18). In this latter study, which focused upon the E. coli replisome as well as
yeast DNA polymerases, Yao et al estimated the frequency of ribonucleotide misincoporation at
one in 2.3 kb during chromosome replication in vivo, a number similar to values reported for
yeast replicative DNA polymerases (19). Studies with yeast replicative polymerases show
varying abilities of polymerases to bypass an incorporated ribonucleotide or to excise it,
depending upon sequence context, the specific polymerase, and the presence of more than one
rNMP in tandem (19, 20, 21). These studies have recently been extended to human polymerase δ
(22).
Several laboratories have shown that ribonuclease H2 plays a predominant role in
initiating excision repair of sites where ribonucleotides have been incorporated (16, 22, 23, 24).
These findings are of great interest for two reasons. First, a genetic deficiency of RNase H2 is
associated with a rare hereditary neuroinflammatory disorder called Aicardi-Goutières syndrome,
a finding that underscores the importance of this enzyme. Second, these findings have led to
identification of the probable mismatch repair system in eukaryotes and in bacteria lacking
methyl-directed mismatch repair. It has long been known that bacteria with the dam methylation
system recognize unmethylated GATC sites to identify daughter DNA strands and correct
mismatches accordingly. How might eukaryotes and prokaryotes lacking the dam system
recognize the daughter strand? Several groups have presented evidence that the presence of nonexcised ribonucleotides in DNA provides just such a marker. Lujan et al (25) have shown that
inactivation of RNase H2 in yeast reduces MMR efficiency preferentially on leading-strand
errors, suggesting that mismatch repair directed by RNase H2 is directed primarily toward
leading-strand replication errors.
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BALANCED dNTP POOL FLUCTUATIONS AS A FIDELITY DETERMINANT
The early interest in dNTP pools as fidelity determinants focused attention upon effects
of unbalanced pools, caused, for example by mutations affecting RNR or dCMP deaminase, as
discussed earlier in this article. An early indication that proportional accumulation of dNTPs
might be mutagenic came when Chabes et al (27) mutated the yeast RNR activity site and found
that dNTP pools expanded by 11- to 17-fold during DNA damaging treatment, with a
disproportionate increase in mutation rates. They proposed that mutagenesis resulted from
elevated dNTP concentrations in vivo, which permitted efficient translesion DNA synthesis.
A different, probably complementary, explanation came from work in our laboratory, not
involving DNA damage as a fidelity determinant (28). In our study dNTP pools in E. coli
expanded near-uniformly as a result of engineered RNR overexpression. Mutation frequencies
increased out of proportion to the pool expansions. Because DNA polymerase-catalyzed chain
extension from a mismatched 3' terminal nucleotide has a much higher KM than does extension
from a matched terminus (29), we proposed that as intracellular dNTP concentrations increase,
the ratio of incorrect to correct chain extension increases. In essence polymerase-catalyzed
proofreading is inhibited because the rate of mismatch extension increases at high [dNTP] with
no significant effect upon the rate of extension from a correctly matched 3' terminus. Since then
at least two other studies, one in yeast (30) and the other in E. coli (31), have found elevated
dNTP pools to be correlated with increased mutagenesis. In both studies the authors describe the
pool expansion as a response to DNA damage; such responses include activation of
ribonucleotide reductase. To the extent studied, it appears that uniform dNTP accumulation is
mutagenic whether or not DNA damage is involved.
If proportional dNTP accumulation stimulates mutagenesis, what about dNTP depletion?
Laureti et al (32) reported a correlation between reduction of dNTP pools and enhanced DNA
replication fidelity. However, these investigators induced the dNTP depletion by disruption of
energy metabolism resulting in ATP depletion, and they did not investigate other possible
consequences of ATP depletion that might also have an effect on mutagenesis. Indeed, an
opposite conclusion was reached by Bester et al (33), who inactivated genes that are normally
up-regulated in carcinogenesis. This led to dNTP depletion accompanied by genomic instability.
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Clearly, to the extent that mutagenesis is related to carcinogenesis, it is important to better
understand the parameters that relate intracellular dNTP concentrations to mutagenesis.
dNTP POOLS AND REPLICATION RATE CONTROL
Are DNA replication rates subject to substrate-level control? A variety of studies suggest
not. Average estimated intracellular dNTP concentrations are in the 5-to-35 µM range (reviewed
in 34), and studies with synchronized cells show that dNTP pool sizes fluctuate dramatically
during the cell cycle, with maximal values, as expected, during S phase. KM values for
eukaryotic DNA polymerases are low; for example, Dresler et al (35) reported that DNA
synthesis in permeabilized mammalian cells proceeded with KM values in the 1- to 3-µM range.
These and other data suggested that average dNTP concentrations within eukaryotic cell nuclei
are severalfold higher than polymerase KM values, and hence, that replisomes operate at substrate
saturation or close to it. The situation is different in prokaryotic systems (36), but it appears that
their replisomes also function at Vmax.
The question whether eukaryotic replication proceeds at Vmax with respect to dNTP
concentration has received critical attention only recently. Poli et al (37) increased intracellular
dNTP pools in budding yeast, either by overexpressing the gene for the RNR R1 protein or by
blocking expression of the RNR-inhibitory protein Mec1. In both cases the rate of DNA chain
elongation, as measured by a DNA-combing assay, was increased, implying that, at least in this
organism, dNTP pools are rate-limiting for DNA replication. However, the relationship is
complex, at least in yeast. Chabes and Stillman (38) found that greater increases in dNTP pools,
10- to 35-fold, also brought about by stimulating RNR expression, overrode the DNA damage
checkpoint and inhibited cell cycle progression. The relationships between dNTP metabolism,
DNA damage, and cell cycle progression comprise a broad field that lies beyond the scope of
this article. The question whether dNTP concentrations during S phase are rate-limiting for DNA
replication merits critical attention.
REGULATION OF dNTP POOLS BY CONTROLLED NUCLEOTIDE DEGRADATION
Most of our early understanding of regulation of metabolite pool sizes focused upon
control of biosynthetic reactions, partly because anabolic regulation appeared to make metabolic
sense and partly because the earliest-investigated allosteric enzymes catalyzed biosynthetic
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reactions. In deoxyribonucleotide metabolism the mechanistic and regulatory complexity of
ribonucleotide reductase focused attention upon dNTP synthesis as the primary site of regulatory
mechanisms. Still, several lines of evidence, primarily in Peter Reichard's laboratory, pointed to
dNTP degradation as an important site for regulating dNTP pools (see 39 for a review of early
evidence and 40 for a more recent review). Reichard and colleagues proposed that dNTP pools
are controlled by substrate cycles, the interconversion of deoxyribonucleoside mono- and
triphosphates. The problem in solidifying this reasonable concept was identifying specific
enzymes in dNTP degradation and describing how their activities are regulated, and how this
regulation might contribute to controlling dNTP pools.
The regulatory importance of dNTP breakdown became much clearer in 2011 with the
simultaneous discovery in two laboratories of an enzyme, SAMHD1, that regulates dNTP pools
by selectively degrading them to the respective deoxyriboncleoside plus tripolyphosphate (41,
42). This protein (sterile alpha motif and HD domain-containing protein 1) was identified as a
protein that restricts lentivirus replication in some myeloid cells and also as a component of the
innate immune response. Moreover, genetic deficiency of this protein in humans was known to
be associated with a form of Aicardi-Goutières syndrome, previously mentioned in this article as
a consequence of RNase H2 deficiency.
Powell et al (41) and Goldstone et al (42) both reported that SAMDH1 is a dNTP
triphosphohydrolase, comparable to the bacterial dGTP triphosphohydrolase mentioned earlier in
this article, which cleaves dNTPs to the corresponding deoxyribonucleoside plus
tripolyphosphate. Both laboratories reported that activity of the enzyme is stimulated by dGTP,
raising the interesting situation that the same molecule, dGTP, is both a substrate and an
allosteric modifier of the enzyme. However, the enzyme is also stimulated by ribo-GTP, which is
present in much higher concentrations, a factor that led Amie et al (43) to propose that GTP is
the true allosteric activator. This latter group also showed that downregulation of SAMDH1
decreased sensitivity of HIV-1-infected cells in macrophages but not in T-cells, by increasing
dNTP levels (44), and also they identified the end-gap repair process in viral integration as the
sensitive step in HIV-1-infected human macrophages (45).
The dNTPase activity explained how SAMDH1 could restrict lentivirus growth—simply
by depleting dNTP pools below the level required for viral reverse transcription (45). Some
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viruses, notably HIV-2, fight back. These viruses encode a protein, Vpx, which targets SAMHD1
for degradation and prevents it from depleting dNTP pools (41)
Franzolin et al (46) have shown that the enzyme plays a broader metabolic role than
simply guarding blood cells against virus infection. These investigators found the enzyme to be
widely distributed in mammalian cells and tissues. Treatment of cells with short inhibitory RNAs
directed at SAMDH1 caused dNTP pools to expand. Variable expression of SAMHD1 during the
mammalian cell cycle supported the idea that the enzyme helps to keep dNTP levels low during
G1. Hence, these findings indicate that SAMDH1 contributes to the control of dNTP levels in
most cells, possibly to the same extent as ribonucleotide reductase.
The SAMDH1 protein was originally described as a homodimer (42), but subsequent
investigation has revealed that inactive monomeric and dimeric forms of the protein tetramerize
in the presence of dGTP (47, 48, 49). Ji et al (48) showed that two molecules of dGTP bind at
each allosteric site. dGTP binding and tetramerization force a conformational change that
facilitates binding of all dNTP substrates at the catalytic site.
An interesting comparison has been drawn to bacterial dNTP triphosphohydrolases,
particularly the enzyme from Enterococcus faecalis, with which the human enzyme shares 36
percent sequence identity (50). The bacterial enzyme is a homotetramer, even in the absence of
effectors. It contains two different classes of binding sites. dGTP binding at the primary site
apparently acts as an “on-off switch,” while the secondary site can bind dNTPs that serve either
as activators or inhibitors of the hydrolysis of specific dNTPs at the catalytic site. The interesting
question how this system, comparable in complexity to rNDP reductase, acts to regulate dNTP
pools in these bacteria, and why, remains to be explored.
An SAMDH1-independent pathway for counteracting HIV-1 infection has recently been
described (51). Thus pathway acts at the level of dNTP synthesis, not breakdown. In this study
the cyclin-dependent kinase inhibitor p21 was found to down-regulate expression of the RNR R2
protein, by repressing the E2F1 transcription factor, thereby inhibiting transcription of the RNR2
gene.
A recent paper (52) describes another protein, DCTPP1, which is suggested to be
involved in controlling dNTP pools. This enzyme is described as a dCTPase, cleaving dCTP to
dCMP and pyrophosphate. Knockout of this enzyme in human cells caused pyrimidine dNTP
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pools to expand. Because data on dATP and dGTP pools were not included in the study, the
function of this enzyme in regulating dNTP pools is not clear.
OXIDIZED dNTPs AND GENOMIC STABILITY
In 1992 the finding (53) that the mutT gene product of E. coli is a nucleoside
triphosphatase focused intense interest upon damaged nucleotides as premutagenic lesions. It is
well known that the major mutagenic target of reactive oxygen species is guanine, which in DNA
undergoes oxidation to 8-oxoguanine. Properties of the E. coli MutT protein and its human
homolog, hMTH-1, suggested that guanine oxidation occurs at the nucleotide level, and that
enzymatic hydrolysis of the oxidized nucleotide, 8-oxodGTP, “cleanses” the DNA precursor
pool by eliminating a damaged nucleotide whose incorporation would be mutagenic.
These early findings stimulated an outpouring of papers supporting this reasonable
conclusion and identifying other modified dNTPs that could similarly represent premutagenic
lesions (reviewed in 54). Most of the approaches used, while persuasive, were indirect, in that
they did not involve actual measurements of intracellular pools of damaged nucleotides—a
difficult task because of their expected low pool sizes. In 2002 our laboratory was frustrated by
our inability to detect 8-oxodGTP accumulation in mutT mutants of E. coli (55); in that study 8oxo-dGTP could have been detected at a minimum intracellular concentration of 0.3 µM.
However, several years later, and with the use of a more sensitive detector, we were able
to find and quantitate 8-oxo-dGTP in extracts of mitochondria from several tissues of the rat. We
looked in mitochondria because of the highly oxidizing conditions that exist in this organelle. In
collaboration with Thomas Kunkel's laboratory (56) we found that the estimated molar 8-oxodGTP concentrations within mitochondria were sufficient to have strong mutagenic effects, as
determined by use of an in vitro replication error detection system. And that led us to wonder
about a puzzling finding from our laboratory (57). Although, as noted earlier, dGTP is almost
invariably the least abundant component of the dNTP pool, typically representing five mole
percent or less of total dNTP, that ratio is reversed in extracts of rat tissue mitochondria, where
dGTP may represent 90 percent or more of total dNTP (58; also see Table I). Although we and
others have not seen such profound dNTP asymmetry in cultured cells (59), and others have not
detected dGTP excess in mouse mitochondria (60), the high dGTP pool in rat tissue
mitochondria is a consistent finding in our laboratory.
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What purpose might this dGTP accumulation serve? One speculative explanation is that it
serves to “buffer” the biological consequences of dGTP oxidation, by mixing 8-oxodGTP with
the much larger dGTP pool and reducing proportionately the incorporation of the damaged
nucleotide into DNA. Readers of this article who can think of straightforward experiments to test
this hypothesis are encouraged to contact the author.
dNTP METABOLISM, CELL CYCLE CONTROL, AND ONCOGENES
There is a growing body of literature describing interrelationships between dNTP pool
regulation and the action of protooncogenes and cell cycle regulators. This is a challenging area
to review, partly because of work done in yeast and mammalian cells, which have similarities but
also distinctions in their modes of cell cycle regulation, partly because the biochemical actions of
some of the protooncogenes are not yet known, and partly because it is not always clear whether
changes in dNTP pools are causes or effects of regulatory phenomena. What follows is
somewhat disconnected and incomplete, offered primarily to give the reader entré to some of the
relevant literature.
Like multicellular organisms, the budding yeast Saccharomyces cerevisiae has an
elaborate genome integrity checkpoint, which senses replication stress that might lead to DNA
damage and acts to relieve that stress. Part of the checkpoint response acts, via the Mec1-Rad53Dun1 pathway, upon ribonucleotide reductase, to ensure that adequate and balanced dNTP pools
are available (61). The human homologs of Mec1 and Rad53 are upstream regulators of p53.
Dun1 is a protein kinase that acts, either during normal S phase or following DNA damage, to
phosphorylate SMl1, a protein inhibitor of RNR activity, targeting it for proteolysis, and thereby
relieving that inhibition and stimulating dNTP synthesis (62). Phosphorylation of another protein,
Dif1, causes release of two RNR small subunits, Rnr2 and Rnr4, from the nucleus to the
cytoplasm, where they combine with Rnr1, a large subunit, to form an active holoenzyme. Full
activation of the Mec1-Rad53-Dun1 checkpoint after DNA damage causes the dNTP pools to
increase by 6- to 8-fold in mostly balanced fashion. An additional gene, Ixr1, interacts with dun1,
to promote the expression of RNR1 and full activation of ribonucleotide reductase (63).
These findings are somewhat reminiscent of an earlier report (64) that unbalanced
nucleotide pools in human cells, arising as a consequence of DNA damage, triggered p53
activation through the human counterpart to the Mec1-Rad53-Dun1 pathway. However, in that
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study, the pool imbalance was induced by N-(phosphonoacetyl)-L-aspartate, an inhibitor of
pyrimidine ribonucleotide biosynthesis. Hence, it was not possible to ascribe the results
specifically to abormalities in deoxyribonucleotide pools.
As discussed earlier, abnormal dNTP pool ratios are highly mutagenic. To what extent
does the genome integrity checkpoint act to recognize unbalanced pools and minimize
mutagenesis? To answer this question Kumar et al (65) generated several RNR loop 2 mutations
affecting the specificity site, as mentioned earlier in this article (2). These mutant strains had
elevated mutagenic potential. The checkpoint responded when one or more dNTPs were depleted,
but not when all four pools were adequate, even if unbalanced. Hence, the consequences of
highly unbalanced pools per se are not sensed as sufficiently damaging to activate the checkpoint.
More recent analysis (66) shows that pre-activation of the genome integrity checkpoint increases
tolerance to multiple DNA-damaging agents, a finding that helps explain the development of
resistance, in humans, to certain chemotherapeutic agents.
Our laboratory has been involved in a collaboration with M. Nikiforov on control exerted
by the C-MYC oncogene on dNTP metabolism (67). Down-regulation of C-MYC caused
specific repression of the genes for thymidylate synthase, inosinate dehydrogenase 2, and PRPP
synthetase 2, with corresponding depletion of dNTP pools and cell cycle retardation.
Overexpression of C-MYC had the opposite effects: enhanced expression of the three target
genes and elevation of the dNTP pools. The data suggested that a major action of C-MYC is
regulation of dNTP synthesis, exerted by control of the expression of the three enzymes just
mentioned.
More recently we described evidence supporting a role for dNTP metabolism in
oncogene-induced senescence (68,69). Human fibroblasts undergoing senescence induced by
HRASG12V expression underexpressed both ribonucleotide reductase and thymidylate synthase,
and possessed subnormal dNTP pools. Reciprocally, ectopic coexpression of the two enzymes,
or addition of exogenous deoxyribonucleotides, suppressed DNA damage and senescenceassociated phenotypes.
Another collaboration, between our laboratory and that of K. Huebner (70) suggested a
more specific relationship between thymidine nucleotide metabolism and expression of the FHIT
oncogene. An early genetic alteration in preneoplasia is alteration of chromosome fragile sites,
leading to loss of Fhit protein expression. In several cell lines we found DNA defects
15
accompanying loss of Fhit expression that could be traced to decreased expression of thymidine
kinase 1, with a consequent specific decrease in dTTP pools. The effects could be reversed or
prevented by addition of sufficient exogenous thymidine.
Finally, a relationship has recently been described between RNR levels and expression of
the antiapoptotic protein Bc12 (71), which mediates inhibitory effects on the G1-to-S-phase
transition. Overexpression of Bc12 in transgenic mice promotes development of lymphomas.
The study by Xie et al (71) showed that expression of Bc12 exerts its effects partly by decreasing
RNR activity, with consequent depletion of dNTP pools. At least part of this effect is brought
about by disrupting the interaction between RNR R1 and R2 proteins, necessary to form a
functional holoenzyme.
TELOMERES AND MICROSATELLITE REPEATS
I conclude this article by briefly mentioning two recent papers highlighting intriguing
relationships between dNTP metabolism and other significant cellular processes. Gupta et al (72)
pointed out that telomeric DNA sequences are guanine-rich, while dGTP is, as noted earlier, the
least abundant dNTP. Examining telomere lengths in yeast, these investigators found relatively
modest effects upon telomere length from varying dNTP pool sizes. However, varying the ratios
of dNTPs had major effects. In particular, telomere length was most sensitive to changes in the
relative dGTP concentration. This effect occurred primarily at the level not of rate, but of
processivity. These findings led Gupta et al to suggest that telomerase activity is modulated in
vivo by dGTP levels. The relationships between telomere metabolism and cancer and other
disease states may focus increased attention on the biochemical reasons why the dGTP pool is so
low in most cells.
The other study, primarily a speculation at this stage, pertains to dNTP metabolism and
the generation of trinucleotide repeats (73). It is generally accepted that repeats are generated by
template-primer slippage during DNA replication. Kuzminov (73) speculated, for example, that
replication of a C-rich template could cause localized depletion of dGTP in the vicinity of a
replication fork, a condition that could retard replication, favoring slippage. The question here, of
course, is the rate at which locally depleted dNTP pools can be refilled by inward diffusion to
replication sites. Issues related to rates of intracellular diffusion of metabolites within cells have
16
been of considerable interest in the past (74). Experiments to examine localized pool depletion
with respect to formation of triplet repeats should be of considerable interest.
ACKNOWLEDGMENTS
I thank my students, associates, and collaborators for their contributions to the work
discussed here, and I apologize to colleagues whose work I was not able to cite, owing to space
limitations.
17
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25
FIGURE LEGEND
Figure 1. Pathways of de novo dNTP biosynthesis in mammalian cells. Salvage pathways are not
shown. Each enzyme name is in italics. Allosteric effectors are shown with upward and
downward arrows, denoting activators and inhibitors, respectively.
TABLE I
Estimated dNTP Concentrations in Mitochondria from Rat Tissues. Data are from reference 56.
Tissue
Estimated Intramitochondrial Concentration µM
dATP
dTTP
dCTP
dGTP
8-oxodGTP
Liver
1.7
1.7
3.9
12.1
1.2
Heart
2.1
3.2
5.6
69.3
1.5
Brain
3.5
0.5
2.8
39.0
0.4
Skeletal muscle
1.6
1.6
4.5
28.4
0.2
Kidney
2.4
3.3
5.7
69.0
1.7
26
de novo synthesis
IMP
AMP
GMP
ATP
ADP
dATP↓
dGTP↑
dADP
UMP
ATP
ATP
GDP
UTP
ATP
Glutamine, ATP
CTP
CTP synthetase
CTP↓ GTP↑
CMP
UDP
dATP↓
dGTP↓
dTTP↑
CDP
dATP↓
rNDP
dTTP↓ reductase
ATP↑
dATP↓
dTTP↓
ATP↑
dGDP
dUDP
dCDP
dCDP
ATP
NDP kinase
ATP
NDP kinase
ATP
NDP kinase
ATP
NDP kinase
dCMP
dUTP
dCMP deaminase
dCTP↑ dTTP↓
H 2O
dUTPase
dUMP
methylene-THF
dTMP
synthase
DHF
dTMP
ATP
dTMP kinase
dTDP
ATP
NDP kinase
dATP
dGTP
Figure 1
dTTP
dCTP