Summary Balanced pools of deoxyribonucleoside triphosphates (dNTPs) are essential for DNA replication to occur with maximum fidelity. Conditions that create biased dNTP pools stimulate mutagenesis, as well as other phenomena, such as recombinationor cell death. In this essay we consider the effective dNTP concentrationsat replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication. We focus upon two questions. (1) In prokaryotic systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that do not equilibrate with the bulk dNTP pools in the cell. Since these pools cannot be analyzed directly, what indirect approaches can illuminate the nature of these replication-active pools? (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide. Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle. To what extent might these natural asymmetries contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome? Introduct ion Of all the chemical reactions that have been studied in living systems. DNA replication is by far the most accurate, with error fre uencies varying in different organisms between lo-’ and per nucleotide replicated(’). If replication fidelity were determined solely by the relative stability of Watson-Crick base pairs, replicative error rates would be far hi her, of the order of lop2 to lop4 per nucleotideo. 4 Several mechanisms contribute toward the greatly increased accuracy of biological DNA replication, including 3’exonucleolytic pr~ofreading(~). kinetic barriers to DNA chain extension from a mismatch(4),and postreplicative mismatch repaid5). Recent years have brought realization that DNA precursor concentrations at replication sites represent an additional determinant of replication accuracy. However, carlier thinking focused upon deoxyribonucleoside triphosphate (dNTP) concentrations more as determinants of replication rate than accuracy. In

prokaryotic cells average dNTP concentrations are subsaturating for replicative DNA polymerases (E.C. 2.7.7.7), yet replication rates seem not to be limited by substrate concentrations in ~ i v o ( ~Attempts ). to reconcile this apparent paradox have led to the roposal, for which considerable evidence now e ~ i s t s ( ~that f dNTP synthesis is physically linked to the replication apparatus, so that dNTP concentration gradients can be maintained at replication sites. Thus, replicative polymerases may sense a small set of dNTP pools, whose levels are high enough to saturate these polyinerases. The situation in eukaryotic cells is still incompletely understood. Some evidence supports the existence of replication-active dNTP pools. However, eukaryotic DNA replication occurs in the nucleus, while dNTPs are synthesized in the cytoplasm(8). Thereforc, if replication-active dNTP pools exist, different compartmentation mechanisms must be involved. For more than a decade it has been realized that unbalanced dNTP levels at replication sites are mutagenic, presumably because they increase the frequency of replication errors(””). Two types of observation led to this realization (both reviewed in references 10 and 11). First, mammalian cells with certain mutations affecting enzymes of dNTP synthesis exhibit both unbalanced dNTP pools and mutator phenotypes, i.e., increased spontaneous mutation frequencies at all loci tested. Second, the deliberate imposition of dNTP pool biases in cell-free replication systems. created from purified replication proteins, was also found to be mutagenic, when the product DNA could be analyzed genetically. Analysis of the early data suggested at least two dNTP concentration-dependent steps in replication: (l}competition between correctly and incorrectly base-paired dNTPs at the insertion step, and (2) the ’next-nucleotide effect’, in which competition between polymerase and 3 ’-exonuclease activities after a misinsertion event could lead to incorporation of a correctly base-paired nucleotide immediately 3’ to a misincorporated nucleotide, before the incorrect nucleotide had been removed by the proofreading nuclease. Subsequent sequence analysis of mutant DNAs produced either in vivo or in v i m confirmed that both of the above mechanisms contribute toward pool imbalance-induced rnutagenesis. However, additional mechanisms must be involved, such as variable efficiencies of proofreading or mismatch repair of specific mismatches, base sequence-dependent effects, or variable effects of dNTP levels on repair of spontaneous DNA damage. All of these possible mechanisms have been reviewed recently(l0>l1) and will not be further discussed here. Rather, we consider the effective dNTP concentrations at replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication. Our analysis will focus upon two distinct but interrelated topics. (1) In prokaryotic

systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that are not equilibrated with the bulk dNTP pools in the cell. Since these pools cannot be analyzed directly, we summarize genetic approaches undertaken in our laboratory, which support the existence of replicationactive pools. (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide. Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle. From known mechanisms by which dNTP pool imbalances contribute toward mutagenesis, we speculate that these natural asymmetries may contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome. Replication-Active dNTP Pools As noted earlier, T4 phage-infected E. coli cells contain a multienzyme complex consisting of phage- and hostcoded enzymes of dNTP biosynthesis, and there is evidence supporting the existence of such a 'dNTP synthetase' complex in uninfected bacteria. We originally thought of this complex as part of a substrate shuttle, which is juxtaposed with the replication apparatus and helps to maintain high local dNTP concentrations at replication sites in the face of rapid turnover('2). Such a model suggests the existence of two kinetically separated dNTP pools. One, a 'replicationactive' pool, would consist of those nucleotides synthesized near replication sites, whose proximity to DNA polymerase would greatly facilitate their utilization for DNA replication. Because of the relatively small number of replication sites (about 60 in a T4infected cell), this pool would be small relative to the 'bulk pool', which is synthesized far from replication sites, is used primarily for DNA repair, and turns over slowly. While it is difficult to determine this latter value, we have estimated(13) that the pools of dNTPs near replication sites are completely turned over ten times per second. Although we still lack direct evidence for physical interactions between dNTP synthetase and the T4 replisome, several lines of evidence suggest that DNA replication, both of T4 and of E. coli, is supplied by dNTPs from pools too small to be measured biochemically(")). Perhaps most persuasive is the observation that rapid DNA synthesis occurs in permeabilized T4infected bacteria provided with deoxyribonucleoside monophosp hates, even though dNTPs cannot be detected in these cells(15).We visualize the replicationactive and -inactive pools as separated kinetically, not physically. Apparently some mixing can occur, because dNTPs that accumulate during a period of reversibly arrested DNA replication (and presumably diffuse away from replication sites) are readil incorporated into DNA once replication is restored . Recently we havc turned to genetic approaches to

gain further insight into the existence and/or nature of replication-active dNTP pools in T4 DNA synthesis. The general approach at first was to impose defined perturbations of dNTP biosynthesis and observe the effects upon rII mutations known to revert to the wildtype genotype along defined mutational pathways(''). In early experiments we asked whether functioning of the dKTP synthetase corn lex in vivo contributed toward replication fidelity('* . We infected E. coli with T4 mutants unable to induce ribonucleotide rcductase (E.C. 1.17.4.1), conditions that prevent the dNTP synthetase complex from forming(7),although all of the other enzymes are present within the cell. At the same time we used an E. coli host strain that overproduces bacterial ribonucleotide reductase, so that there would be no limitation of DNA synthesis rates imposed by precursor supply. Under these conditions, AT-to-GC and GC-to-AT substitution mutations were both increased by small but reproducible factors - two-fold to five-fold. While these observations shed no direct light on the nature of replication-active dNTP pools, they do suggest that the dNTP synthetase complex plays a role in maintaining normally high replication fidelity. Somewhat more specific information was obtained when we used the same r l l reversion approach to investigate the mutator phenotype of certain temperature-sensitive mutations in T4 gene 42(13). Gene 42 encodes deoxycytidylate hydroxymethylase (E.C. 2.1.2.b), an enzyme that catalyzes the following reaction:

P '

dCMP

+ 5,10-methylene-H4folate + 5-hydroxyniethyl-dCMP

+ H4folate

This enzyme is principally responsible, along with an enzyme that hydrolyzes dCTP to dCMP, for the substitution in T-even phage DNAs of 5-hydroxymethyl-cytosine for cytosine. Amber and ts gene 42 mutants display a replication-defective phenotype under nonpermissive conditions, because they cannot accumulate pools of hydroxymethyl-dCTP, while the dCTP pool is also being depleted. However, at semipermissive temperatures, c.g., 34". some fs gene 42 mutants of T4 reproduce satisfactorily, but the phage resulting from such infections are enriched for mutations in all examined. In further investigating this phenomenoii(l3), we confirmed the finding of Williams and Drake that these conditions preferentially stimulated reversion of rII mutations that proceed along GC+AT pathways. One such mutation, T4 rIISN103, showed an eightyfold increase in level of reversion at 34" as compared to 28". This is what we expect if the effect of the 34" incubation is to partially inactivate dCMP hydroxymethylase, so as to creatc a localized deficiency of hydroxymethyl-dCTP at replication sites. Other nucleotides, notably dTTP, would compete with the shrunken hydroxymethyl-dCTP pool for incorporation opposite template G. The resultant G-T mismatch, if unrepaired, would give an A-T base

100 0

I

I

r l l SN103 reverslon

10

dATP, dTTP pools

a,

m

._ !? c

1.0

m K

0.10

0.01 Temperature 'C

Fig. 1. Temperature sensitivity of biological parameters in E. coli infected with T4 tsLB3, a phage carrying a temperaturesensitive mutation in gene 42. Data are from reference 13.

pair on the next round of replication, completing the GC+AT transition. When we measured the relevant biological parameters (Fig. l), we detected no significant changes in the dNTP pools at 34", when compared to those measured at 28". dCMP hydroxymethylase activity. as measured in extracts, was diminished only slightly at this temperature. Note that at higher temperatures, where DNA synthesis is virtually abolished, with concomitant diminution or dNTP utilization, dATP and dTTP both accumulated, as previously shown for this and other DNA-defective T4 mutants(20). We interpret the above observations as follows: (1) the replication-active dNTP pools are extraordinarily sensitive to even partial inhibition of pathways leading to their synthesis, because of their extremely high rates of turnover; (2) the resultant depletion of hydroxymethyl-dCTP at replication sites increases mutation rates because its decreased concentration relative to other dNTPs improves the ability of the latter to compete for incorporation opposite template G; (3) the replication-activc pool constitutes but a minute fraction of the total dNTP within a T4-infected cell, because the mutagenic responses observed occur in the absence of

biochemically detectable changes in total intracellular dNTP content. To test this concept further, we determined the n ucleotide sequences of several wild-type and pseudowild revertants from rirlSN103. Only one of eleven such strains sequenced was a true revertant, with a GC-AT transition occurring at thc site of the original mutation. Five more were pseudorevertants, each of which underwent a GC-AT transition at the adjacent base pair. The remaining five involved GC-TA transversions, either at the site of the original mutation or at the adjacent site. We hypothesize that these transversions occur by formation of a mutagenic G-A base pair, with dATP competing with hydroxymethyl-dCTP for incorporation opposite template G. The main point is that all of the mutations analyzed could well have arisen as the consequence of a hydroxymethyl-dCTP pool depletion, biochemically undetectable because it affected only the small replication-active pools of DNA precursors. Investigations of m utagenesis that involve reversion as the biological end-point are limited, because they cannot sample a wide variety of base sequence contexts. The existence of hot spots., or sites of preferential mutagcnesis, for most types of replication errors indicates that neighboring sequences exert a significant influence on error rates. Such base sequence effects are clearly determinants of mutagcnesis caused by perturbations of dNTP metabolism. For example , conditions that impose a hydroxymethyl-dCTP accumulation stimulate r l l reversion mutations that occur by AT+GC pathways(17). While most such reversions were stimulated about ten-fold, one such mutation, rIIUV215, was stimulatcd to revert by more than one thousand-fold. In order to sample a wide variety of sequence contexts, what one nccds for analysis is a forward mutation assay system, where one can readily select mutations occurring at many sites on the gene and determine the sequence at these sites. Sequence analysis of many such revertants leads to identification of hot spots, where the same mutational event occurs repeatedly. Examining base sequences surrounding such hot spots can generate insights regarding mutagenic mechanisms. We have found that the T4 tk gene, which encodes thymidine kinase (E.C. 2.7.2.2), presents a suitable forward mutation assay systcm. While the coding sequencc for this enzyme, 588 nucleotides, is fairly long, the gene is amenable to repeated analysis in an automated sequencer. More importantly, tk- mutants can be readily selected by growth of a lysate in the presence of 5-broniodeoxyuridine, followed by nearUV irradiation of progeny phages(21).Wild-type phage incorporate BrdUrd into their DNA, which causes radiosensitization, whereas tk- mutants are enriched among the survivors of such a regimen because they cannot incorporate BrdUrd. In a continuing analysis(22),we have isolated 16 tkmutants from separate lysates of a ts gene 42 mutator

T T T T

5'-

T

Yc- 1c-cT

t

1

c-c-c-cc-

3'

T

I

T

100

C-3

C

T T TAT 200

a

T T T

300

4 00

500

588

T4 tk gene nucleotide position

Fig. 2. Mutagcncsis induced by action of a thcrmolabile dCMP hydroxymethylase. Mutations induced in thc T4 tk gene by growth of T4 fsLB3 at 34", semi-permissive but mutagenic conditions. LMost of these mutations were sequenced by JJ and reported in his Ph.D. thesis, but some mutants were isolated and sequenced in our laboratory by Jiazhi Sun.

mutant, grown at 34". Results from sequence analysis of the mutant tk genes are shown in Figure 2. Note that mutations are distributed throughout the coding region. The 16 mutations analyzed to date include twelve GC-AT transitions and two GC-TA transversions, in accord with the concept that a localized hydroxymethyldCTP deficiency causes increased substitution of dTTP or dATP opposite temple G, just as seen in analysis of the rII revertants. However, the forward mutation data are potentially more informative. Note that the sixteen sequenced mutations occurred at just fourteen sites; two of the sites are represented by two mutations each. Since all of the mutants were isolated independently, this suggests the existence, even among a small collection of mutants, of hot spots. The entire mutational target includes 199 Cs (actually HMC, or hydroxymethylcytosine). Even if substitutions at some of these sites don't generate a mutant phenotype, the existing data suggest a clustering of mutant sites, which might, after analysis of a larger mutant collection, reveal sequence context features favorable to mutational events. For now, however, we limit ourselves to the conclusion that the forward mutation data support the concept of a small, replication-active hydroxymethyldCTP pool, which can be significantly depleted in the absence of detectable changes in the total intracellular dNTP contents. While this concept is consistent with the physically coupled dNTP synthesis-DNA replication model, direct biochemical evidence is still required.

dNTP Pool Asymmetry and Variable Genome Evolut ion In this section we explore the question whether natural asymmetries in dNTP concentrations at replication sites contribute toward variable spontaneous mutation frequencies in different genes within the same cell. The traditional view of molecular evolution holds that point mutations occur at uniform rates throughout the genome of an organism. and that variations in rates of nucleotide substitution among different genes in a

genome reflect selection mechanisms, which favor retention of some but not all mutations. However, Wolfe, Sharp, and Li(23) presented evidence that different regions of a mammalian genome undergo variation at different rates, even in the absence of selective pressure. They compared DNA sequences of homologous genes from closely related organisms (humans vs. primates, rats vs. mice), with attention focused specifically upon nucleotides at four-fold degenerate sites. These sites could undergo any substitution mutation without affecting the amino acid sequence of the gene product. Even though this criterion should rule out effects on variation based upon either positive or negative selection, they found that the frequency of such variations (a parameter called K4) varied by at least three-fold among several dozen genes analyzed. K4 values for these expressed genes were comparable to rates of variation in non-expressed regions of the genome (pseudogenes and intergenic regions), sequences where any mutations should be truly neutral. Thus, even in the apparent absence of selective pressure, significant differences were seen in the rates at which different genes underwent mutation in the same genome. (Note that this analysis does not directly address the question of fihether spontaneous mutations that confer a selective advanta e occur more frequently than truly neutral mutations(F. 2h).) The analysis of Wolfe el al. showed the rate of gene sequence variation to be a function of the guanine+cytosine content of the four-fold degenerate sites. Although there was considerable scatter in the data points, it appeared that I(4values were highest in genes containing about SO% G + C in the four-fold degenerate sites, with reduced variation rates on either side of this 50% value. In attempting to rationalize their observations, Wolfe et al. cited data from our laboratory("), which showed that dNTP levels within mammalian cell nuclei undergo significant changes as synchronized cell cultures proceed through S phasc of the cell cycle. In particular, the relative G + C content of the dNTP pool increased with time. Since relative dNTP concentrations affect replication fidelity('-''), and since genes are replicated at specific times in the cell cycle("), Wolfe et al. proposed that the rate of spontaneous mutagenesis varied with time during S phase, such that the rate of sequence variation of a gene would depend upon the time at which that ene was replicated. In a subsequent analysis, Wolfe(2 has presented a model (with the help of some simplifying assumptions) suggesting that the only conditions under which G + C content of a gene would not influence spontaneous mutagenesis are those involving equimolar dNTP pools and equal dNMP mole fractions in DNA. While it is true that we demonstrated significant dNTP pool changes during S phase, the data that we published provide only partial support for the authors' hypothesis. First, our observed changes in G+C content of the dNTP pool were relatively small - from about 44 percent of the total dNTPs at the end of G1

8

phase to about 55 percent in the middle of S phase(27). These changes are comparable to those observed by others in analyses of whole-cell dNTP pools during synchronous growth of mammalian cells. Second, our data were obtained with Chinese hamster ovary (CHO) cells, since these cells can easily be synchronized in quantity by isoleucine deprivation, a treatment that does not directly perturb dNTP pools. Some CHO cell lines, however, have very low levels of dCMP deaminase (E.C. 3.5.4.12), and as a consequence, they have unusually high dCTP pools when compared to the primate, mouse, and rat cells analyzed by Wolfe et al. Therefore, at this stage one cannot conclude that the magnitude of dNTP pool changes during S phase is sufficient to account for the apparent differences noted in replication fidelity, or even that the varying rates of gene evolution do result from variations in replication fidelity. Clearly. data arc needed on cell cycle variations of intranuclear dNTP levels in other mammalian cell lines. Such data may be difficult to obtain, first, because the small sizes of nuclear relative to whole-cell dNTP pools necessitate large amounts of cells for reliable measurements, and second, because most synchronization methods adaptable to large culture sizes involve deoxyribonucleotide pool perturbations. However, such data are essential, a5 shown in part by the following. In a recent study, we compared two thy- mutator strains of CHO cells, which display quite different elevations of spontaneous mutation frequencies(”). We were able to explain the basis for these differences in terms of deoxyribonucleotide pool abnormalities only when we analyzed dNTP pools in nuclei of synchronized S-phase-enriched cells. Thus, these latter measurements reflect the dNTP environments sensed by the replication machinery more faithfully than do other existing measurements of dNTP pool size or distribution. Despite our limited knowledge of intranuclear dNTP changes during S phase, one can propose mechanisms to account for variations in spontaneous mutation frequency based upon G + C content of the gene, even in the absence of dNTP pool fluctuations. In mammalian cells, dGTP is almost always the least abundant of the four common dNTPs, as measured in whole-cell extracts. Figure 3 summarizes data from several laboratories. The limited data available, from our laboratory, confirm this asymmetry for intranuclear dNTP pool sizes. If one assumes that effective dGTP concentrations are subsaturating for replicative DNA polymerases in v i m , then at least two factors can contribute to replication accuracy as a function of G+C content in template DNA. While the two mechanisms would have opposite effects upon replication error frequency as a function of G + C content, they could explain why error frequencies might be maximal in a template with about SO percent G+C. First, substitution errors opposite dCMP residues in the template would be enhanced, because of competition between dGTP. the ‘correct’ nucleotide, and the

A human

= hamster

mouse

dATP

I -

8 0

.

AAAAAAAU A

dCTP 8 .

-

A

8

m

a

A - A

0 0

n

Y

AM-

I

A dGTP

. I

A

m

A

0

20

W

.

8

AAAAAAAA

40

dTTP 8

AA

60

80

100

% of total dNTP uool

Fig. 3. dNTP pools in extracts of mammalian cells - human, mouse, and hamster, as indicated. A cell with equimolar dNTP pools would show each of the four deoxyribonucleotides at 25% of the total dNTP pool. Data are compiled from reference 27 and 31-41. Data from dCMP deaminase-deficient cell lines are omitted.

far more abundant ‘incorrect’ dNTPs (dATP in the example shown).

3-l

C

C

Mispairing effect

This factor would tend to increase mutation frequency as a function of increased G + C content in the template. Since most of the data in Figure 3 identify dCTP as the second least abundant nucleotide, substitution errors opposite template G might also be enhanced, contributing further toward decreased accuracy of replication of GC-rich DNA. Second, exonucleolytic proofreading could be enhanced at sites on the immediate 5’ side of dCMP in the template strand. If dGTP levels are subsaturating, then the residence time of 3’-terminal dNMPs just upstream from such sites would be increased because of the decreased rate of insertion of dGMP opposite dCMP in the template. This ‘next-nucleotide effect’

would tend to increase replication accuracy as a function of increased GC content of the template. 3’- GC S-T

-GC-

TdTMP

-dCTP

dGTP

Next-nucleotide effect

-GC-CG

-

content and global mutation frequency will be a complex function of the relative activities of polymerases and proofreading nucleases and the effective concentrations of the four dNTPs in viim. Adding to the complexity are observations suggesting highly variable efficiencies of mismatch error correction systems in mammalian cells, with respect to specific mismatched base pairs(46). The above factors make it virtually impossible to predict precise relationships between template G + C content and mutation rates. However, in vitro systems are available for asking whether (1) pool asymmetries com arable to those seen in mammalian cells and nuclei (2) cell cycle-dependent changes in pool an&, asymmetries can account for enough replication errors to explain the observed rates of evolutionary variation within a single genome. Such investigations are the focus of current efforts in our laboratory.

For the above factor to contribute to replication accuracy, the effective dGTP concentration in vivo must be below the optimal concentration for replicative DNA polymerases. While it is difficult to determine the exact molar concentration of a metabolite within an organelle, it is likely that dGTP levels are subsaturating. Of course, even if one docs know with accuracy both the number of dGTP molecules per nucleus and the volume of the nucleus. one cannot conclude that intranuclear dGTP has an effective concentration equal Acknowledgements to that of an equimolar dGTY solution in a typical biological buffer. Despite this uncertainty, we estiResearch in our laboratory is supported by NSF mated the intranuclear dGTP pool in S-phase CHO cell Research Grant DMB8916366 and NIH Research nuclei to be about 10 PM(’~).In human cells, KMvalues Grant GM37508. We thank Eric Anderson, a graduate for replicative DNA polymerases, whether measured student in CKM’s course in Nucleotide Biochemistry, with purified enzymes or permeabilized cells, are in the for bringing Reference 23 to our attention and for 1-5 pM range(4-), suggesting that dGTP levels are suggesting an experimental test of the model developed slightly below optimum values. Note, however, that the in the latter half of this article. Data in the first half of kinetic data pertain to extension from correctly basethe article are from a Ph.D. thesis submitted by JJ, who paired 3’ termini. Extension from mismatched termini is now located in the Department of Pathology. is kinetically discriminated against. For example, University of Washington, Seattle, WA 98195. Mendelman et al.(43)reported KM values ranging from 300 to 1000 ,uM for extension from a mismatch, strongly References suggesting that the next-nucleotide effect, as described above. does contribute toward replication fidelity in a I Drake, J. W. (1991). A constant ratc of spontaneous mutation in DNA-habed microbes. Proc. Natl Acad. Sci. U S A 88, 7160-71M. dNTP concentration-dependent fashion. Therefore, 2 Loeh, L. (1974). Errors in DNA replication ar a basis of malignant changes. dGTP levels are almost certainly subsaturating in vivo Cancer Res. 34. 2311-2321. 3 Kunkel, T. A. (1988). Exonucleolytic proofreading. Cell 53, 837-840. for extension from a mismatch, as are levels of all four 4 Uunlin, M. J., Patel, S. S. and Johnson. K. A. (1991). Kinetic partitioning dNTPs. between the esonuclease and polymerase sites in DNA error correction. As noted earlier, dNTP pool imbalances are well Biuchemi\ try 30, 5.q-546. 5 Modrich, P. (1987). DNA mismatch correction. A n n u . Rev. Biochem. 56. known to be mutagenic, both in vivo and during 435-466. replicative DNA synthesis in vilro. Both incorporation 6 Mathews, C. K. and Sinha, N. K. (1982). Are DNA precursors concentrated errors and next-nucleotide effects are i n v o l ~ e d ( ’ ~ ” ~ ~ )at. replication sites? Proc. Natl Acrid. Sci. USA 79, 302-306. 7 Mathews, C. K., Moen, L. K., Wang, Y. and Sargent, R. G. (1988). Are the dNTP asymmetries in vivo, as determined from Intracellular organization of deoxyribonucleotide-synthesizing enzymes. pool measurements, large enough to influence mutation Trends in Biochem. Sci. 13, 394-397. 8 Mathews, C. K. ‘and Slahaugh, M. B. (1986). Eukaryotic DNA replication: rates? Very likely, yes. In studies on the fidelity of Are precursors channeled to replication sites’! Exptl. Cell Res. 162, 285-29.5. replication in vitro, catalyzed by human DNA polym9 Fersht, A. R. (1979). Fidelity of replication of phage qx174 by DNA erases, Kunkel and his colleagues routinely impose a polymerase I11 holoenzyme: Spontaneous mutation by misincorporation. Proc. Natl Acad. Sci. USA 76, 496-4950, twenty-fold bias, and these asymmetric dNTP mixtures 10 Meuth, M. (1989). The molecular b of mutations induced by do stimulate mutagenesidG); a fivefold 001 bias was deoxyribonucleoside triphosphate pool imbalances in mammalian cclls. Expfl. Cell Kes. 181, 305-316. mutagenic in a sensitive reversion assay$’). Biases of 11 Kunz, B. A. and Kohalmi, S . E. (1991). Modulation of inutagcnesis by comparable magnitude are readily seen among the data deoxyribonucleotide levels. A n n u . Rev. Genet. 25. 339-359. compiled in Figure 3. Thus, it is likely that pool 12 Reddy, G. P. V., Stafford, M. E., Singh. A. and Mathews, C. K. (1977). Enzyme associations in T4 phage DNA prccursor biosynthesis. Proc. Natl asymmetries in vivo contribute toward determining Acad. Sci. 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Christopher K. Mathews and Jiuping Ji are a t the Department of Biochemistry and Biophysics, Oregon State University, Cowallis, Oregon 97331-6503. USA.