Use of aurintricarboxylic acid as an inhibitor of nucleases during

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-

Nucleic Acids Research

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.I

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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Use of aurintricarboxylic acid as an inhibitor of nucleases during