T H E J O U R N A I .

OY B I O L O G I C A L CHEMISTRY
Vol 465. No. 14. Issue of duly 25, pp 6640-6645, 1980
Printed m C!S A

Detection ofActivities that Interfere with the EnzymaticAssay of

Deoxyribonucleoside 5’-Triphosphates*
(Received for publication, February 1, 1980)

Thomas W. North, Richard K. Bestwick, and Christopher K. Mathews

From the Department of Biochemistry and Biophysics, Oregon State University, Coruallis, Oregon 97331

Several enzymes that interfere with the enzy- aration and analysis ( 2 )and equilibrium labeling of pools with
matic assay of deoxyribonucleoside B’-triphosphates :12P(11).The HPLC methodis sensitive only down to 30 pmol
(dNTF”s)are present as contaminants when nucleotides (2), while ”tP-labeling procedures require long term labeling
are extracted fromHeLa cellswith 60% methanol. of cells and complex chromatographic separations. The .’lP
These activities include a nuclease, nucleoside diphos- method is also unsuitable for tissues.Therefore, theenzymatic
phokinase, and deoxyribonucleoside monophosphoki- procedure seems more suitable for rapid, sensitive assays of
nases which phosphorylate dAMP,dGMP, and dCMP. cells containing low levels of dNTP’s.
Collectively, these enzymes are able to degrade and In order to measure dNTP levels by any of these procedures,
reutilize the DNA template which is used together with the nucleotides must be efficiently extracted from cells. This
DNA polymerase for dNTP assays. This process intro-
extraction mustbe rapid so that cellular enzymes do not alter
duces large errors when dNTP assays are performed in
this manner. dNTP levels during the process, either by degradation or by
Attempts to block the enzymatic conversion of de- continued synthesis of dNTP’s. With the enzymatic assay,
oxyribonucleoside diphosphates to triphosphates by such cellular activities mustalso be completely removed from
inhibition of nucleoside diphosphokinase were unsuc- extracted samples to prevent t.heir interference during the
cessful because of the inability to block completely the assay reaction.
kinase activity. Several procedures for extracting nucleotide pools have
Acid extraction of nucleotides also resultsin the pres- been reported. The most commonly used include extraction
ence of an activity that interferes with the enzymatic with 60% methanol (4) or with acid. Acid-extraction proce-
dNTP assay. The error introduced by this interfering dures use perchloric acid (6), acidic ammonium formate (12),
activity is much smaller than that arising from the or trichloroacetic acid (13).
enzymes present in methanol extracts. All of these We havenoted somelargedifferences in dNTP levels
interfering activities are removed when cells are first measured in different laboratories on similar biological mate-
extracted with 60%methanol and the resulting extract rials. For example, in herpes simplex virus-infected cells, the
is subsequently treated with perchloric acid. levels of dCTP were reported to be more than 50-fold higher
by Jamieson and Bjursell (7) and by Roller and Cohen ( 8 ) ,
both of which groups used 60% methanol extraction, than by
Previous reports from this and other laboratories (reviewed Cheng et al. ( 6 ) ,who extracted with perchloric acid. Since all
in Ref. 1 ) have supported the existence of amultienzyme of these reportsused a similar enzymaticdNTP assay,we felt
complex containing the enzymes of DNA precursor biosyn- that the discrepancies were due to differences in extraction
thesis which suppliesdeoxyribonucleoside 5”triphosphates procedures. Different extraction efficiencies or thepresence of
during replication of bacteriophage T4. In aneffort to deter- interfering activities in any of the extracts could produce the
mine whether similar enzyme complexes exist in eukaryotic observed variation.
cells, we are attempting to determine the subcellular locali- For our studies DNA on precursor metabolism, it is essential
zation of dNTP’sl and the enzymes involved in their synthesis. that we accurately quantitate levels of these molecules. Ac-
Intracellular levels of dNTP’s are much lower than are cordingly, we have evaluated the enzymatic assay of dNTP’s
use of assays using various methods to extract nucleotides. We have en-
levels of ribonucleotides ( 2 ) .This necessitates the
withsensitivitiesin the picomole rangetoquantitatethe countered interfering activities in these assays, which are
dNTP levels in cultured cells. An enzymatic assay for dNTP’s reported in this paper.
with a sensitivity down to 1 pmol has been developed (3, 4) EXPERIMENTAL PROCEDURES’
and used widely ( c i Refs. 5-10). In this assay, the level of a
RESULTS
particular dNTP is determined by measuring the incorpora-
tion of radioactively labeled counternucleotide into a defined Properties of the dNTP assay with nucleotidestandards are
DNA template(usuallyanalternatingcopolymer) in the presented under “Supplemental Data” and in Fig. 1 in the
presence of DNA polymerase. Other methods that havebeen miniprint.
developed include high pressure liquid chromatographic sep- d N T P Assays of Standards Added to Extracts-When
dTTP or dATP standards were added to 60% methanol-ex-
* This work was supported by Research Grant CA-24323 from the
National Institutes of Health. The costsof publication of this article ’’ Portions of thispaper(including“ExperimentalProcedures,”

were defrayed in part by the payment of page charges. This article
musttherefore be herebymarked“aduertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
’ The abbreviations used are: dNMP, dNDP, and dNTP,deoxyri-
bonucleoside 5’-mono-, di-, and triphosphates, respectively; HPLC,
high pressure liquid chromatography.
“Supplemental Data,” and Figs. 1 to 7) are presented in miniprint at
the end of thispaper.Miniprintiseasilyreadwiththeaidofa
standard magnifying glass. Full size photocopies are available from
the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda,
Md. 20014. Request Document No.0-207, cite authors, and include a
check or money order for $1.35 per set of photocopies.

6640

This is an Open Access article under the CC BY license.

 Enzymatic Assay of d N T P S 664 1
tractedpreparations from HeLa cells, they were detected in mitochondrial preparations (Fig. 4B).The extractsused for
quantitatively with this assay (Fig. 2). The activity measured the experiment in Fig. 4A degrade ,"H-labeled DNA at a rate
after addition of standard to extract was equal to the sumof >20 ng/min/106 cells (data not shown).
individual activities of standard and extract. Thus, nothing in Degradation and reutilization of the template by the com-
theseextractsinterferes with theenzymaticdetection of bined activities of a nuclease, dNMP kinases, and nucleoside
dNTP's. diphosphokinase could result inincreased levels of dATP,
Surprisingly,when we addeddNDPstandardstothese dGTP, and dCTP as measured with this assay (but not in
extracts, their activities were identical with equimolar addi- increased dTTP levels because no dTMP kinase is present).
tons of dNTP standards (Fig. 2). In addition, we found that Indeed, we have observed that apparent levels of dATP and
["HldTDP is converted to ["HIdTTP and incorporated into dCTP (asmeasured with this assay) aremore than five times
DNA under these conditions (data not shown). The level of higher in 60% methanol-extracted HeLa cells than in prepa-
activity with these added dNDP's is much higher than ex- rations from extraction with ammonium formate orperchloric
pected from the reaction with dNDP standards (Fig. 1) and acid. Levels of dGTP and dTTP are comparablein all three
suggests the presence of nucleoside diphosphokinase activity preparations. The reason that dGTP levels are not elevated
in the 60% methanol extracts. in assays of the methanol extracts is that the poly(d1-dC)
Addition of dTMP standards to these methanol extracts templatecannot be recycled to produce dGTP. When
resulted in no increase in apparent dTTP levels (Fig. 2 A ) . poly(dG-dC) wasused in thesereactions,apparentdGTP
However, addition of dAMP standardsincreased the apparent levels were much higher in the methanol extracts thanin the
dATP levels of these extracts (Fig. 2B). This activitywas not other extracts (data not shown).
seen in the assay of dAMP standardsin the absenceof extract Thus, treatment of cells with methanol extracts not only
(Fig. 1) and thus appears to be due to dAMPkinase as well as dNTP's, but also enzymes that interfere with the enzymatic
nucleoside diphosphokinase in these extracts. dAMP kinase assay of dNTP's. The above data support a role for these
activity of 5 to 10 pmol/min/106 cells in these extracts was enzymes in producing artifactually high values for dNTP's
estimated from the amount of apparent dATP detected from when these procedures are used.
theaddeddAMP(Fig. 2B). Similar levels of dGMPand Other Extraction Procedures-An interfering activity was
dCMP kinases were detected in this manner (datanot shown). also detected in acid extracts. This activity prevented the
Kinase Activities in Extracted Nucleotide Preparations- enzymaticassayfromreaching plateau values. Details are
Direct assay of 60% methanol extracts for nucleoside diphos- presented under "Supplemental Data" in the miniprint.
phokinase activity confEms the presence of this activity (Ta- Elimination of Interfering Activities by a Two-step Pro-
ble I). This activity (which is stable to freeze-thawing, lyoph- cedure-It is clear from the above data that methanol extrac-
ilization, and heating to 65°C for 20 min) represents 30% of tion leads to serious overestimation of dNTP pools, and that
thetotalHeLa cell nucleoside diphosphokinase(Table I). acid extraction cannot be used because of the failure of acid
This enzyme was not detected in extracts prepared by two extractstoreachplateau values. However, the interfering
acid-extraction procedures (TableI). activities in methanol extracts aredifferent from those in acid
The levels of dNMP kinase activities that were estimated extracts. Therefore, we felt that a combination of these two
from the above data are below the level of detection by our extraction procedures might eliminate dl interfering factors.
spectrophotometric assay. As expected, therefore,we detected Accordingly, we extracted cells with acidic methanol (0.5 N
no dAMP kinase activity by direct assay of 60% methanol perchloric acid in 60% methanol). The dATP assaysof these
extracts. We also failed todetectthymidylatesynthetase extracts (Fig. 6) gave results identical with those from per-
activity in these extracts. chloric acid extracts (Fig. 5 ) , without alleviating the interfer-
Attempts to block the enzymatic conversion of dNDP's to ence.
dNTP's with desdanine, an inhibitor of nucleoside diphos- However, we were able to eliminateall of these interfering
phokinase, were unsuccessful due to the inability to inhibit activities by combining these two extraction methods in a
completely the kinase activity. Details of these experiments two-step procedure. Cells were first extractedwith 60% meth-
are presented in the miniprint. anol and the extracts were taken to dryness. This material
Nuclease Actiuity-A nuclease activity is alsoobserved was subsequentlyre-extracted with 0.5 N perchloricacid.
during dNTP assaysof material extracted from cells with 60% When these extracts were assayed for dATP, the timecourse
methanol. This activity is detected only in concentrated prep- was identical with that obtained with dATP standards (Fig.
arations where apparent dNTP levels are well above the linear 6). The reactionswere also complete by 60 min and the level
range of the reaction. With these extracts, "H-labeled dNTP of radioactivity incorporated remained constant thereafterin
which is incorporated into DNA during the initial period of assays of these extracts for dGTP, dCTP, and dTTP (data
the reaction is later released (Fig. 4A). This activity decreases not shown).
as the extract is diluted, isand not detectedin assays of d N T P The amount of each dNTP detected in these extracts was
standards (Fig. 4A). In our attempts to measure mitochondrial directly proportional to the amount of extract used for the
dNTP levels, we noted that this activity is particularly high assay (Fig. 7). Moreover, known amounts of dNTP's added to
these extracts were detected with an accuracy of 93 to 110%;
TABLE I "H-labeled dNTP's added to cells and carried through the
Nucleoside diphosphokinase actiuities extraction were recovered with greater than 95% efficiency
Enzyme source Actiwty (data not shown). Thus, thelevel of dNTP's present in these
extracts can be accurately determined with the enzymatic
HeLa cell lysate 5240 nmol/rnin/l0" cells
60% Methanol extract 1610 nmol/rnin/lO" cells
assay.
Ammonium formate extract (5 nmol/rnin/lo" cells The levels of the four dNTP's determinedfrom HeLa cells
Perchloric acid extract t 5 nmol/min/lO" cells extracted in this manner are shown in Table 11. The values
E.coli DNA polymerase I 0.13 prnol/min/unit" which we obtained from several different HeLa cell prepara-
' A unit of E . coli DNA polymerase I is the activity which incor- tions were very consistent and ranged from 64 to 76 pmol/106
porates 10 nmol of total nucleotide into an acid-precipitable product cells and 78 to 100 pmol/106 cells for dATP and dTTP,
with activated DNA as template.primer in 30 min a t 37°C. respectively.
6642 Enzymatic Assay of d N T P s
TABLEI1 The cell extracts used in these studieswere from HeLacells.
Determination of dNTP levels in HeLa cell extracts Similar results were obtained with Chinese hamster ovary
Extracts were prepared by the two-step procedure described in the
cells and with sea urchin eggs (data not shown). It appears
text. that these interfering activities are a general problem in the
dNTP assay of biological materials.
prnol/lb' cells"
We have eliminated all of these interfering activities by
dATP 74
extracting cells with 60% methanol and then treating this
dTTP 82 extract with 0.5 N perchloric acid. The enzymatic assay of
dGTP 9.8 these extractsis similar to the assayof dNTP standards, and
dCTP 31 standard dNTP's are accurately measured when added to
" These values are averages of determinations from three different these extracts. In addition, the measured dNTP levels are
extracts for dATP and dTTP, and from two different extracts for proportional to the amountof extract. Moreover, nucleotides
dCTP and dGTP. are stable to these extraction procedures. Therefore, these
methods are superior to existing methods for measurement of
dNTP pools.
DISCUSSION Since enzymes or interfering activities from other cells may
In order to understand the control and regulation of DNA vary in their sensitivities to these extractions, control experi-
precursor biosynthesis and the relationshipof this process to ments similar to those reported here will benecessary to
DNA replication, accuratemeasurements of intracellular determine the suitability of these methods for dNTP assays
dNTP levels are necessary. Quantitativedetermination of of other cells, tissues, and organelles. When nonenzymatic
dNTP levels is also important in evaluating effects of certain assays are used, these activities may not interfere. However,
antimetabolites, particularly nucleoside analogs and anti-fol- because of the substantial levels of enzymes present in meth-
ate compounds. Moreover, certain metabolic disorders, such anol extracts, special care must be taken to ascertain that no
as combined immunodeficiency disease which is due to the breakdown or interconversion of nucleotides occurs during
absence of adenosine deaminase, produce alterations in dNTP handling and assay of samples.
levels (20, 21). Accurate quantitative determinationof dNTP The dNTPlevels we have determinedin HeLa cells are less
pools is necessary todeterminethe involvement of these than 20% of the values reported by others who have used
alterations in the course of the disease. methanol extraction and the enzymaticassay (7,8, 10). When
Most published data on dNTP pools were obtained with we assay methanol extracts,we obtain similar high valuesand
the enzymatic assay for dNTP's, and material was extracted attribute them to the interfering activities we have described.
by one of the procedures investigated in this study. With theseValues reported from enzymatic assay of perchloric acid ex-
methods, we have found several activities that interferewith tracts (6) are comparable to levels we have determined, in
thisassayandmightproduceerrors in measurement of agreement with our observation that the interfering activity
dNTP's. First, highly purified Escherichia coli DNA polym- of these extractscauses amuch smaller error than that arising
erase I contains nucleoside diphosphokinase activity, which from methanol extracts.
results in an overestimate of dNTP levels when dNDP's are The levels of dATP and dTTPwhich we have determined
present. Since this activity is low and dNDPlevels are usually are in close agreement with those reported by Garrett and
much lower than dNTP levels, the error introduced in this Santi (2) who have developeda HPLC method. However,
manner should be small. Nevertheless, akinase-free DNA their values fordCTP and dGTP are two to three times higher
polymerase is necessary to obtain a high degree of accuracy. than ours. We have not determined whether this is due to the
We have found that bacteriophage T4 DNA polymerase is use of different cells (they used mouse lymphoma cells) or to
free of nucleoside diphosphokinase, but it will not efficiently a difference in the methods. Our dNTP determinations are
catalyze the reaction at the low levels of d N T P required in also comparable to values obtained with a :'2P-labeling tech-
these assays. nique (11).
A more formidable problem is the presence of interfering We have found that the intracellular level of d C T P is
activities inextracts obtainedby common methods for extrac- substantially lower than levels of the other three dNTP's.
tion of nucleotides. Extraction with 60% methanol results in This was also observed by Garrett and Santi (2) and others.
preparations containing a nuclease, dNMP kinases, and very The dGTP pool is similarly lower in prokaryotic systems,
high levels of nucleoside diphosphokinase. Collectively, these namely E. coli and bacteriophage T4-infected E. coli (12). It
enzymes(alongwith DNA polymeraseused in theassay) will be of interest to determine whether dGTP levels are
break down and reutilize the DNA template during the en- limiting for HeLa cell DNA synthesis.
zymatic assay for dNTP's. These enzymes are not present in We are currently using these methods to determine the
two acid-extracted nucleotide preparations (0.5 N perchloric subcellular location of DNA precursors.
acid or 0.4 N ammonium formate, pH3.0).However, the time Acknowledgments-We thank Gerald W . Lasser and Dr. James R.
course of the reactionwith acid extracts is different from that Allen who performed some of the enzyme assays, and Ginnie L. T.
with standard dNTP's, which precludes accurate determina- Moffett for excellent technical assistance.
tions in these extracts. REFERENCES
The presence of these interfering activities, particularly the 1. Mathews, C . K., North, T. W., and Reddy, G. P. v. (1979) A h .
high level of enzymes in 60% methanol-extracted material, Enzyme Re&. 17, 133-156
mayexplain the large variabilityin dNTP levels reported 2. Garrett, C . , and Santi, D. V. (1979) Anal. Biochem. 99,268-273
from different laboratories thatuse similar biological systems 3. Lindberg, U., and Skoog, L. (1970) Anal. Biochem. 34, 152-160
(see above).Generally,values reported from methanol-ex- 4. Skoog, L. (1970) Eur. J . Bzochem. 17,202-208
tracted samples are much higher than those extracted by 5. Skoog, L., Nordenskjold, B. A., and Bjursell, K. G . (1973) Ew. J .
Biochem. 33,428-432
other procedures. 6. Cheng, Y.-C., Goz, B., and Prusoff, W . H. (1975) Biochim. Bio-
phys. Acta 390,253-263
,'T. W. North, R. K. Bestwick, and C. K. Mathews, unpub- 7. Jamieson, A. T., and Bjursell, G. (1976) J. Gen. viral. 31, 101-
lished results. 113
 AssayEnzymatic
8. Holler, B., and Cohen, G. H. (1976) J. Virol. 18, 58-64
9. Mathews, C. K. (1975) Exp. Cell Res. 92, 47-56
10. Hellgren, D. Nilsson, S., and Reichard, P. (1979) Biochem. Bio-
phys. Res. Commun.88, 16-22
11. Reynolds, E. C., Harris, A. W., and Finch, L. R. (1979) Biochim.
Biophys. Acta 561, 110-123
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77,446-463
14. North, T. W., and Cohen, S. S. (9178) Proc. Natl. Acad. Sei. U.
S. A. 75,4684-4688
15. Murray, R. E., and Mathews, C. K. (1969) J . Mul. Biol. 44, 233-
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of dNTP’s 6643
16. Mathews, C. K. (1966) Biochemistry 5, 2092-2100
17. Duckworth, D. H., and Bessman,M.J. (1967) J . Bcol. Chem. 242,
2877-2885
18. Miller, L. K., and Wells, R. D. (1971) Proc. Natl. Acad. Sci. U. S.
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20. Cohen, A,, Hirschhorn, R., Horowitz, S. D., Hubinstein, A,, Pol-
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1626
6644 Enzymatic Assay of dNTPS

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