Use of aurintricarboxylic acid as an inhibitor of nucleases during ...
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Volume 4 Number 9 September 1977 Nucleic Acids Research
Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation
Richard B. Hallick, Barry K. Cheim, Patrick W. Gray and Emil M. Orozco, Jr.
Department of Chemistry, University of Colorado, Boulder, CO 80309, USA
Received 3 June 1977
ABSTRACT
Aurintricarboxylic acid (ATA) is a general inhibitor ofnucleases. ATA has been shown to inhibit the following en-zymes in vitro: DNAse I, RNAse A, Sl nuclease, exonucleaseIII, and restriction endonucleases Sal I, Bam HI, Pst I andSma I. The observed inhibition is consistent with the pro-posal by Blumenthal and Landers (BBRC 55, 680, 1973) that mostnucleic acid binding proteins will be sensitive to ATA. Theaction of ATA as a nuclease inhibitor can be used to advantagein the isolation of cellular nucleic acids.
INTRODUCTION
The triphenylmethane dye aurintricarboxylic acid (ATA) isknown to inhibit a number of enzymatic reactions of proteinand nucleic acid biosynthesis. ATA inhibits both the initia-tion and elongation of protein synthesis (1-5), the in vitroreactions of QS replicase, E. coli DNA-dependent RNA poly-merase and T7 RNA polynmerase (6), viral RNA-directed DNA poly-merase (7), chloroplast RNA polymerase (8), and the ATPase
activity of E. coli RNA synthesis termination factor p (9).Blumenthal and Landers (6) have predicted that ATA will inhi-bit most, if not all, proteins that bind nucleic acids, pre-
sumably as a competitive inhibitor at the polynucleotidebinding site. Recently ATA has been shown by Bina-Stein andTritton (10) to inhibit not only enzymes of polynucleotidemetalolism, but also many other enzymes regardless of their
catalytic function.Since ATA is a potent inhibitor of most nucleic acid
binding enzymes, we reasoned that it might serve as a useful
general nuclease inhibitor to prevent degradation of nucleicacids by endogenous nucleases during isolation. Data are
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England 3055-
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presented on the inhibition of a number of.nucleases in vitro
at low ATA concentration, and on the use of ATA to inhibit
nucleases during the isolation of cellular RNA and DNA.
MATERIALS
ATA, Aluminon grade, from Synthetical Laboratories, was
used without further purification. Pancreatic DNAse I, pan-
creatic RNAse A, calf thymus DNA, and yeast RNA were purchased
from Worthington Biochemicals. E. coli exonuclease III was
from New England Biolabs. Restriction endonuclease Sma I was
generously provided by Iqor Dawid.
METHODS
RNAse A activity was determined by the method of Kalnitskyet al. (11). DNAse I activity was measured by the procedure
of Kunitz (12). Sl nuclease was isolated by the method of
Sutton (13). The activity of Sl nuclease in digesting l H]-
E. coli DNA was measured as previously described (14). Re-
striction endonuclease Pst I was isolated by the method of
Smith et al. (15). X DNA was isolated by published procedures,
(16). Isolation of restriction endonucleases Bam HI, EcoRl
and Sal I, assay of endonuclease activity, agarose gel electro-
phoresis, and photography of results have been described (17).
Restriction endonuclease reactions were incubated for 3.5
hours at 370.Chloroplasts of Euglena gzracilis were used for RNA and
DNA isolation. ATA (1mM) was added to all buffers. For RNA
isolation, Euglena chloroplasts were isolated by the procedure
of Brawerman and Eisenstadt (18). Purified chloroplasts were
suspended in acetate buffer (0.05 M sodium acetate, 1 mM
ethylenediaminetetraacetate, 0.5% sodium dodecylsulfate, pH 5)
containing 1 mM ATA, and extracted twice with phenol previouslysaturated with acetate buffer. The aqueous layer was next
extracted twice with CHCljisoamylalcohol (24:1; v:v). The RNA
was collected by ethanol precipitation, and redissolved in
0.015 M NaCl, 0.0015 M sodium citrate (0.1 x SSC). For DNA
isolation, covalently closed, superhelical Euglena chloroplastDNA was isolated by centrifugation of chloroplast lysates in
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CsCl-ethidium bromide (17). Residual ATA was removed by pas-
sage of the RNA or DNA solution through a 1 x 30 cm column of
Sephadex G-100 previously equilibrated with 0.1 x SSC. The
ATA irreversibly associated with the gel at the top of the
column.
A 2% agarose, 6M urea gel was used for electrophoreticanalysis of the chloroplast RNA (19). Vertical slab gels, 9cmwide by 7cm long and lmm thick were cast between glass plates.
RNA was electrophoresed for 5 min at 75ma, followed by 90 minat 30ma. Gels were stained for 30 min at 40 in a 5 pg/mlethidium bromide solution and photographed as described above.
RNA was also fractionated on a 0.3-1.4 M linear sucrose gra-
dient in 0.02 M Tris-HCl, 0.1 M NaCl, 1 mM ethylenediamine-
tetraacetate, 0.2% sarkosyl, pH 7.6, at 120,000 g for 24 hrs
at 40 in a Spinco SW 41 rotor.
RESULTS
In vitro inhibition of nucleases by ATA.
The activity of a number of nucleases was tested in thepresence of various concentrations of ATA. Results on the in-
hibition of bovine pancreatic DNAse I and Sl nuclease from
AspergiZZus oryzae are shown in Figure 1. DNAse I is found to
be strongly inhibited by ATA, in agreement with the results of
Bina-Stein and Tritton (10). When the DNA substrate concen-
tration is 33.3 pg/ml, inhibition is complete at 10 pM. Slnuclease is also inhibited by ATA, but at higher concentrations.At 50 pM ATA, Sl nuclease is approximately 50% inhibited. It
is possible that lower ATA concentrations would have given in-hibition if initial reaction velocities had been determined inthe Sl nuclease assays.
Results on the inhibition of bovine pancreatic RNAse A
are shown in Figure 2. RNAse A is strongly inhibited by ATA.
Inhibition is complete at approximately 10 aM when the RNA
substrate concentration is 3.5 pg/ml.ATA is a potent inhibitor of E. coli exonuclease III.
Following incubation of X DNA with a large excess of exo-
nuclease III for 3.5 hours at 37°, no polynucleotides were
detectable in the reaction mixture (Figure 3, lane 2). The
amount of enzyme was more than 25-fold greater than needed
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80 SI 80
H60 ~z60_
U40- U 40-
0~~~~~~~~~~~~~
0 10 20 30 40 50 - 6 1 14 103ATA (x106M) ATA(M)
Figure 1 - (Left) Inhibition of deoxyribonucleases by ATA.The effect of various concentrations of ATA on DNAse I and Slnuclease is illustrated. The DNAse I substrate was calf thy-mus DNA. Activity is expressed as percent of initial reactionvelocity. The Sl nuclease substrate was a mixture of heatdenatured [3H]-E. coZi DNA (0.27 pg/ml) and nonradioactivecalf thymus DNA (10 ig/ml). Sl reactions were incubated 2hours at 50°.
Figure 2 - (Right) Inhibition of RNAse A by ATA. The effectof various concentrations of ATA on the initial reaction velo-city of bovine pancreatic RNAse A is illustrated.
for a limit digest. When 100 pM ATA was added to the above
reaction, exonuclease III activity was inhibited, and highmolecular weight X DNA (>30 kbp) was present in the productmixture after a 3.5 hour reaction (Figure 3, lane 1).
Five restriction endonucleases were found to be completely
inhibited by ATA. As illustrated in Eigure 3 (lanes 4, 6, 8
and 10), digestion of X DNA by Sma I, Sal I, Pst I, or Bam HI
gave the expected limit digestion products (20). When 100
PM ATA was added to the above reaction mixtures, only undi-gested DNA was observed after a 3.5 hour reaction (Figure 3,
lanes 3, 5, 7, 9). Restriction endonuclease EcoRl was also
found to be inhibited by ATA (not shown).From the above results it is seen that ATA inhibits in
vitro a number of nucleases that have different modes of
nucleic acid cleavage. These include endonucleases that act
on single stranded DNA (Sl nuclease), double stranded DNA
(DNAse I), and at specific sites on double stranded DNA
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1 2 3 4 5 6 7 8 9 10
Figure 3 - Inhibition of exonuclease III and restriction en-donucleases by ATA. X DNA (0.68 pg) was treated as follows:(1) exo III + 100 pM ATA (2) exo III (3) Sma I + 100 phi ATA(4) Sma I (5) Sal I + 100 pM ATA (6) Sal I (7) Pst I + 100 pMATA (8) Pst I (9) Bam HI + 100 pM ATA (10) Bam HI. Productswere analyzed by electrophoresis on a 0.7% agarose gel.
(restriction endonucleases). ATA also inhibits an exonuclease
(exonuclease III) and a ribonuclease. All nucleases that we
tested were inhibited by ATA. Although data for only eightenzymes are presented, it seems reasonable to suggest that
ATA is a general nuclease inhibitor.
Use of ATA to inhibit nucleases during RNA isolation.The effect of ATA on the isolation of chloroplast RNA
from chloroplasts of Euglena gracilis was examined. RNA was
chosen for these experiments because it is more sensitive to
enzymatic degradation during isolation than DNA. Isolation of
Euglena chloroplasts involves a typical, lengthy subcellular
fractionation procedure during which there is continuous de-
gradation of nucleic acids by nucleases present in the cell
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lysate. An attempt was made to retard nuclease degradation by
the addition of 1 mM ATA to the buffers used during chloroplast
RNA isolation.
A sucrose gradient sedimentation profile of the chloro-plast RNA isolated in the presence of ATA is shown in Figure
4. The chloroplast rRNAs at 16S and 23S are the prominent
bands (21,22). When ATA was not present during chloroplast
isolation, the resulting profile was altered. The boundary
between the rRNAs was not well defined, and the absorption
profile was skewed toward the low molecular weight range. The
presence of ATA resulted in an increased yield, and better pre-
parative separation of the chloroplast rRNAs.
The chloroplast RNA was also analyzed by gel electro-
phoresis. RNA from chloroplasts isolated in the presence or
absence of 1 tiM ATA are compared (Figure 5). A third RNA
sample, with 1 mM ATA and 50 pg/ml heparin as nuclease in-
hibitors is also shown. Consistent with the sucrose gradientresults, the yield of high molecular weight chloroplast RNA
is greatly enhanced in the (+) ATA samples. There is con-
siderable RNA below the 16S band only in the (-) ATA sample.
The three most intense bands in the (+) ATA samples are the
0.5
0.3-
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0 10 20 30 40FRACTION NO.
Figure 4 - Analysis of isolated Euglena chloroplast RNA bysucrose gradient sedimentation. RNA was isolated either inthe presence (triangles; 63 pg sample) or absence (circles;51 pg sample) of 1 mM ATA. The centrifuge tube was orientedfrom the top, right, to bottom, left.
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Figure 5 - Analysis of chloroplast RNA byelectrophoresis in an agarose/urea gel.Each sample contains 1.5 , g of RNA. Thesamples are from left to right (1) (-) ATA(2) (+) ATA (3) (+) ATA and heparin.Direction of electrophoresis was from topto botton.
16S and 23S rRNAs, and an rRNA aggregate that forms under the
electrophoretic conditions employed. There is a much lower
yield of these components in the (-) ATA RNA. Also signifi-cant are the 7-8 discrete bands between 16S and 23S rRNA in
the (+) ATA samples, and largely absent in the() ATA con-
trol. At least one and perhaps-several of these may representintact chloroplast mRNAs. Following translation of the(+
ATA P14A in an erythrocyte cell free protein synthesis systemthe major p-roduct was a polypeptide that comigrates with the
large subunit of ribulose-1,5-diphosphate carboxylase duringelectrophoresis (B. K. Chelm and R. B. Hallick, unpublishedobservation). This is the expected product (23). No largesubunit polypeptide was evident following translation of the
-)ATA RNA.
Use of ATA to inhibit nucleases during DNA isolation.The effect of ATA on the isolation of chloroplast DNA
from chloroplasts of Euglena gracilis was also examined.Since chloroplast DNA, and DNA from many other sources can beisola'ted in intact form without DNAse inhibitors being pre-
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sent, it is difficult to generalize about the utility of ATA
for DNA preparations. In two different preparations, the
yield of covalently closed, circular DNA from isolated chlo-roplasts was at least as good when ATA was added to the buf-
fers. Furthermore the ATA could be removed from the DNA bygel filtration chromatography, to permit subsequent restric-tion endonuclease digestion experiments. As shown in Figure6, chloroplast DNA isolated in the presence of ATA and
chromatographed on a gel filtration column gave the same EcoRl
digestion pattern as a (-) ATA control DNA. Similar results
were obtained with the restriction enzymes Bam Hl and Sal 1
(not shown). DNA isolated in the presence of ATA was also a
substrate for the nick translation activity of E. coli DNA
polynterase I. In this experiment, (+) ATA chloroplast DNA
could be activated by pancreatic DNAse I, and labeled by DNA
polymerase I with I3HJ-TTP as a substrate to a specific
activity >107 dpm/pg, comparable to previously reported re-
sults with (-) ATA chloroplast DNA (14).
Figure 6 - Restriction endonucleasedigestion of Euglena chloroplast DNAisolated in the presence or absence of ATA.Chloroplast DNA was digested withendonuclease EcoRl. Left, (+) ATADNA; right, (-) ATA DNA.
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DlSCUSSION
We have found ATA to be an effective, general inhibitorof nucleases both in vitro and in the isolation of cellularRNA. As an RNAse inhibitor, ATA may be added to the pre-sently available arsenal that includes bentonite, heparin,diethylpyrocarbonate, and others. ATA may be used eitheralone or in concert with these agents. For some types ofDNA isolations, ATA may also prove to be a useful DNAse inhi-bitor.
Several advantages of ATA as a nuclease inhibitor are
apparent. It is an inexpensive, highly water-soluble compoundthat may be used at concentrations considerably higher thanneeded to inhibit nucleases in vitro. Addition of ATA to
buffers during subcellular fractionation did not interfere
with nucleic acid isolation. ATA is readily removed from
purified nucleic acids by gel filtration chromatography. RNAisolated in the presence of ATA is translatable in a cell freesystem. DNA isolated in the presence of ATA can be digestedwith restriction endonucleases, and can serve as a template-primer for DNA polymerase I. There is no obvious disadvantageto the use of ATA other than the highly colored nature of thesubstance.
ACKNOWqLEDGEMENTSWe gratefully acknowledge the technical assistance of
Hema Sista. This work was supported by Grant GM 21351 fromthe National Institutes of Health.
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