- Email: [email protected]
32P-base analysis of DNA
ANALYTICAL
BIOCHEMISTRY
117, 271-279 (1981)
32P-Base
Analysis
of DNA’
M. VIJAYARAJ REDDY, RAMESH C. GUPTA, AND KURT RANDERATH Department
of Pharmacology,
Baylor
College
of Medicine,
Texas
Medical
Center.
Housron.
Texas
77030
Received March 31, 1981
A “P-labeling method for the base composition analysis of nonradioactive DNA was developed consisting of the digestion of DNA to deoxynucleoside 3’-monophosphates by incubation with a mixture of micr ococcal nuclease and spleen phosphodiesterase, transfer of ?label from [T-‘~P]ATF to the 5’-hydroxyl groups of the mononucleotides by T4 polynucleotide kinase, two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose of the resultant [ 5’-32P]deuxynucleoside 3’,5’-bisphosphates, autoradiography, and scintillation counting. The method was standardized to afford quantitative digestion of DNA to mononucleotides as well as to give quantitative incorporation of ?-label into the nucleotides in the DNA hydrolysate so as to make the method an accurate means for determining the base composition of eucaryotic DNA containing adenine, guanine, thymine, cytosine, and 5-methylcytosine.
The presence in DNA of bases other than adenine, guanine, thymine, and cytosine is a feature common to all organisms. Such modified bases may arise by enzymatic modification of DNA bases (i.e., 5-methylcytosine and N6-methyladenine) or by the action of chemicals (e.g., chemical carcinogens) on DNA. Several methods are available for the identification and quantitation of major and modified constituents of DNA. The earliest technique used for this purpose was paper chromatography (1). Paper and thin-layer chromatography (2) have also been used to analyze DNA that has been labeled in vivo with radioactive isotopes (3,4), an approach not well suited for the analysis of DNA from intact experimental animals and humans. More modern chromatographic techniques applied to this task have included gas chromatography (5), high-performance liquid chromatography (6-l l), and gas chromatography-mass spectrometry ( 12). Mass spectral ( 13) and immunological ( 14- 17)
methods have also been utilized to identify and to determine specific DNA components. Current methods for determining the base compositions and sequences of amounts of nonradioactive nucleic acids which are too small for conventional chromatography entail analysis by postlabeling techniques. Thus, for the base composition and sequence analysis of RNA (18-20) and for the sequence analysis of DNA (21), the nonradioactive nucleic acid is specifically broken down into smaller components which are then labeled with radioisotopes and are characterized by chromatographic or electrophoretic techniques. This approach has been particularly useful for the identification and the quantitation of modified constituents in RNA ( 18), but somewhat surprisingly has thus far not been applied to DNA base composition analysis. Davies et al. (22) recently reported experiments that demonstrate the feasibility of determining the composition of a mixture of the four major deoxynucleoside 3’-monophosphates by a 32P-postlabeling technique, but these authors have not applied this technique to the analysis of DNA.
’ Supported by USPHS grant CA-25590.
271
0003-2697/81/160271-09$02.00/O Copyright 0 198 I by Academic Press, Inc. All rights of reproduction in any form reserved.
REDDY,
272
GUPTA,
This article details a 32P-postlabeling method for base composition analysis of DNA which consists of the enzymatic digestion of DNA to deoxynucleoside 3’monophosphates, conversion of these compounds to [ 5’-32PJdeoxynucleoside 3’,5’bisphosphates by the T4 polynucleotide kinase-catalyzed phosphorylation reaction, separation of the 32P-labeled nucleotides by anion-exchange thin-layer chromatography on PEI-cellulose,’ and quantitation of these nucleotides by scintillation counting. This work has been presented in preliminary form (23). MATERIALS
AND METHODS
Materials
2’-Deoxynucleoside 3’,5’-bisphosphates (PL Biochemicals), 5-methyl-2’-deoxycytidine 5’-phosphate (Sigma Chemical Co. ), NJVbis(2-hydroxyethyl)glycine (Bicine) (Calbiochem), [Y-~~PJATP (Amersham), T4 polynucleotide kinase (P-L Biochemicals), and potato apyrase (grade I, Sigma Chemical Co.) were used without further purification. The hexanucleotide, dATGCAT (Collaborative Research, Inc.), was purified by PEI-cellulose thin-layer chromatography ( 18) before use. Micrococcal nuclease (grade VI, Sigma Chemical Co., 0.21 U/rg) and spleen phosphodiesterase (BoehringerMannheim, 0.002 U/pg) were dialyzed against water at 4°C for 15 h. Female Sprague-Dawley rats weighing 150- 185 g were partially hepatectomized according to the method of Higgins and Anderson (24) and liver DNA was isolated after 24 h by a modification of the procedure of Marmur (25). This and other DNA preparations (Sigma Chemical Co.) were treated with 0.3 N KOH at 37°C for 15 h, neutralized with 1 N HCl, precipitated with alcohol, and dialyzed against water at 4°C for 15 h to remove ribonucleotides. PEI-cellulose sheets (20 x 20 cm) without indicator (Brinkmann 2 Abbreviations Smethylcytosine.
used:
PEI,
polyethyleneimine;
mSC,
AND
RANDERATH
Instruments, Inc.) were prewashed as described (26) prior to use. XAR-5 films (Eastman-Kodak Co.) and Lighting Plus intensifying screens (DuPont) were used for autoradiography. Concentrations of dATGCAT and DNA were estimated by uv absorption at 260 nm at neutral pH using units/mg for dATGCAT (from 29.9 A260nm data sheet of the supplier) and 20 &Onm units/mg for native DNA (27). Model Experiments 32P-labeling of model nucleotide mixture.
An equimolar mixture of dAp, dGp, dCp, and dTp (total concentration, 6 PM), [y32P]ATP (60 PM, 10 Ci/mmol), and T4 polynucleotide kinase (0.25 U/PI) in 10 ~1 of a solution containing 40 mM Bicine-NaOH, 10 mM MgC12, 10 mM dithiothreitol, and 0.1 mM spermidine, pH 9.0, was incubated at 37°C for 60 min. Control reactions were conducted without the addition of the nucleotides, but were further processed like samples containing the nucleotides. A 2-~1 aliquot of the 32P-labeled reaction mixture was added to 8 ~1 of a solution containing 30 mrvr sodium periodate, 50 mM sodium acetate, and 12 mM EDTA, pH 5.0, and incubated at 22°C for 10 min. After the addition of 2 ~1 of a solution containing 4 hg each of dpAp, dpGp, dpCp, and dpTp, the mixture was applied to a PEI-cellulose sheet (10 X 20 cm) at a point 2.5 cm from both the left-hand and bottom edges. Development in the first (short) dimension was with water to the origin, followed by 1.5 M LiCl to the top (-7.5 cm from the origin). The sheet was dried, soaked in 500 ml of methanol with gentle shaking for 10 min, and dried. For the second (long) dimension, chromatography was with 1.75 M ammonium formate, pH 3.5,3 to 2 cm on a Whatman No. 1 wick attached to the top of the sheet (- 17.5 cm from the origin). Individual ’ This prepared hydroxide.
and other ammonium by titration of formic
formate solutions were acid with ammonium
‘*P-BASE
ANALYSIS
nucleotide spots were located by screen-intensified autoradiography (28), cut out, and their radioactivity was determined by Cerenkov counting. Equivalent areas were cut from control chromatograms and were counted. These blanks were subtracted from the sample values. Base ratios were calculated directly from the corrected values and were expressed as percentages of the total. Studies with dATGCAT. The compound (1 pg) was hydrolyzed to deoxynucleoside 3’-monophosphates with 2 rg of spleen phosphodiesterase in 10 ~1 of 20 mM sodium succinate, pH 6.0, at 37°C for 2 h. Control reactions were conducted without the addition of dATGCAT and were further processed like samples containing nucleotides. The nucleotides in the digest were 32P-labeled, chromatographed after treatment with periodate, and assayed as described above. Standardization of Conditions for DNA Base Analysis DNA digestion. In these experiments, which were designed to optimize the digestion conditions, either enzyme:substrate ratios or digestion times were varied. In the first set of experiments, 1 pg of DNA was digested with varying amounts of a 1: 1 mixture of micrococcal nuclease and spleen phosphodiesterase at 37°C for 2 h in 10 ~1 of a solution containing 20 mrvt sodium succinate and 8 mM CaCl,, pH 6.0. In the second set of experiments, 1 pg of DNA was digested at 37°C for different time intervals with 2 pg each of micrococcal nuclease and spleen phosphodiesterase in 10 ~1 of the same buffer. The nucleotides in the digests were 32P-labeled and chromatographed, and their radioactivity was determined as described above. 32P-labeling of nucleotides in DNA digests. To standardize the labeling conditions,
DNA was digested to deoxynucleoside 3’monophosphates which were 32P-labeled and chromatographed as described in the following section except that the 32P-labeling re-
OF
DNA
273
action was conducted for various intervals of time, and additional T4 polynucleotide kinase (final, 0.85 U/pi) was added at 180 min. Standard Procedure for Quantitative Analysis of DNA
Base
Digestion and “P-labeling. For quantitative digestion of DNA to deoxynucleoside 3’-monophosphates, 1 pg of DNA (- 3 nmol of DNA-P) was incubated at 37°C for 2 h with 2 pg each of micrococcal nuclease and spleen phosphodiesterase in 10 ~1 of a solution containing 20 mrvr sodium succinate and 8 mM CaC12, pH 6.0. Control reactions were conducted without DNA and were further processed like the samples containing nucleotides. The digest was diluted IO-fold and a 2-J aliquot containing -60 pmol of deoxynucleoside 3’-monophosphates was mixed with a solution of [T-~*P]ATP (60 PM, 1O-20 Ci/mmol) and T4 polynucleotide kinase (0.25 U/pi) in 10 ~1 of a solution containing 40 mM Bicine-NaOH, 10 mM MgC12, 10 mM dithiothreitol, and 0.1 mM spermidine, pH 9.0. Incubation was at 37°C for 1 h. Chromatography and quantitative analysis. The 32P-labeled mixture (10 ~1) was
incubated with 2.5 cclof a solution of potato apyrase (5 mU/pl) at 37°C for 1 h to convert excess [ T-~*P]ATP and 32P-labeled impurities (see Results and Discussion) to 32Pi before chromatography. Aliquots (2 ~1) of the reaction mixture were applied to each of 4 to 6 PEI-cellulose sheets (20 X 20 cm) which had been soaked in 500 ml of 0.1 M ammonium formate, pH 3.5, for 30 min and dried, first in a current of cool air, then warm air (5 min each). The sheets were developed overnight, at least 15 h, in 0.8 M ammonium formate, pH 3.5, to about 25 cm on a Whatman No. 1 wick which had been attached to the top of the sheet by stapling and then had been folded. Development was then continued for 2 to 3 h in 4 M ammonium formate, pH 3.5, without intermediate drying.
REDDY,
214
GUPTA,
The sheets were dried as above, soaked in 250 ml of methanol for 10 min, and dried briefly in cool air. For the second dimension, chromatography was with water to the origin, followed by 0.3 M ammonium sulfate to 4 to 5 cm on a Whatman No. 1 wick attached to the top of the sheet. Individual nucleotide spots were located by autoradiography, cut from the chromatogram, and their radioactivity was determined by Cerenkov counting. Base ratios were calculated, after subtracting blank values, from the 4 to 6 replicate analyses as described above.
AND
RANDERATH
j2P-Labeling of model nucleotide mixture. Davies et al. (22) have reported that
FIG. 1. Two-dimensional separation of a “P-labeled digest of dATGCAT on a PEI-cellulose thin-layer sheet. The deoxynucleoside 3’-monophosphates obtained by enzymatic digestion of the hexanucleotide were “P-labeled and chromatographed as described under Materials and Methods. The specific activity of [y-‘*P]ATP was 10 Ci/mmol. After NaI04 treatment, the ‘*P-labeled digest of dATGCAT (total radioactivity, 1.3 &i; 12 pmol of deoxynucleoside 3’,.5’-bisphosphates) was applied to the chromatogram. Development in the first dimension (from bottom to top) was with 1.5 M LiCI, and in the second dimension (from left to right) with 1.75 M ammonium formate, pH 3.5. Autoradiography was at -70°C for 11 h.
a mixture of dAp, dGp, dCp, and dTp (total concentration, 6 PM) could be quantitatively phosphorylated by incubation with 60 I.LM [ Y-~*P]ATP and 0.06 U/p1 of T4 polynucleotide kinase at pH 9.2 and 37°C for 30 min as assayed by DEAE-paper electrophoresis and counting. Using the conditions described under Materials and Methods, we also found the phosphorylation of deoxynucleoside 3’monophosphates to proceed to completion. These conditions, which are slightly different from those given by Davies et al. (22), have been adapted to give optimal phosphorylation rates with a commercial preparation of T4 polynucleotide kinase. In addition, our procedure specifies ion-exchange thin-layer chromatography to separate the 32P-labeled nucleotides (see Fig. 1) because this technique affords better separations of modified nucleotides than does DEAE-paper electrophoresis (29). Before chromatography, the mixture of 32P-labeled nucleotides was treated with sodium periodate to convert excess [Y-~*P]ATP to the dialdehyde, most of which was retained at the origin by binding to the amino groups of polyethyleneimine during subsequent chromatography. Studies with dATGCAT. Davies et al. (22) have shown that the base composition
of a ribooligonucleotide could be determined by enzymatic digestion to ribonucleoside 3’monophosphates and enzymatic 32P-labeling. These authors did not report an extension of this approach to deoxyoligonucleotides or DNA. We have chosen dATGCAT as a model compound to test the accuracy and precision of the “P-labeling method for the determination of the base composition of a deoxyoligonucleotide. This oligonucleotide was digested to deoxynucleoside 3’monophosphates and the products of the 32Plabeling reaction were chromatographed after treatment of the reaction mixture with sodium periodate as described under Materials and Methods. The resolution of ‘*P-labeled deoxynucleoside 3’,5’-bisphosphates is shown in Fig. 1. Individual ‘*P-labeled compounds were identified by cochromatography with authentic markers, and their radioactivity was determined. In addition to 32P-labeled nucleotides and 32Pi,several other spots contributed by [T-~*P]ATP were visible after prolonged exposure (Fig. 1). The bulk of [T-~*P]ATP was retained at the origin. The expected base ratio of A:G:C:T for this compound is 2:l: 1: 1 since thymidine derived
RESULTS AND DISCUSSION
Model Experiments
“P-BASE
ANALYSIS
from the 3’-terminus by spleen phosphodiesterase digestion is not a substrate of the polynucleotide kinase reaction. The average base ratio calculated from 8 determinations was 2.02 (+0.057):1.01 (f0.051):0.94 ( IL 0.040): 1.04 ( It_0.033). Standardization of conditions for DNA analysis. Subsequently, conditions were
standardized for the digestion and base composition analysis of DNA containing the four major bases and m5C. The accuracy of these determinations is dependent on obtaining quantitative hydrolysis of DNA and quantitative labeling of the digestion products. We have chosen a mixture of micrococcal nuclease and spleen phosphodiesterase for the digestion of DNA because the former enzyme digests DNA to a mixture of deoxynucleoside 3’-monophosphates and dinucleotides carrying S-hydroxyls and 3’phosphates (30) while the latter enzyme digests oligonucleotides carrying 5’-hydroxyls to nucleoside 3’-monophosphates (3 1). Thus, a combination of the two enzymes was expected to afford quantitative hydrolysis of DNA to the deoxynucleoside 3’-monophosphate level.
16
-
ro 12 k? x6E . Et4 I
T C A G I 0.01
I
11111110 0.1
0.03
1 * 111.111 0.3 I
1
I
2
Micrococcal Nuclease + Spleen Phosphodiesterase (pg/ul each) FIG. 2. DNA digestion with varying amounts of a 1:l mixture of micrococcal nuclease and spleen phosphodiesterase. Regenerating rat liver DNA (0.1 pg/pl) was digested to deoxynucleoside 3’-monophosphates which were “P-labeled, chromatographed, and assayed as described under Materials and Methods. Chromatography in LiCl/ammonium formate solvents (see Materials and Methods) did not separate dpm’Cp and dpCp; thus C denotes the sum of dpCp and dpm’Cp. T denotes dpTp; G, dpGp; and A, dpAp.
OF
DNA
275
DNA digestion was studied by varying enzyme and DNA concentrations. The digests were subjected to quantitative ‘*P-labeling and chromatographic analyses. The results obtained by digesting DNA (0. I pg/ ~1) from regenerating rat liver with varying amounts of micrococcal nuclease and spleen phosphodiesterase at 37°C for 2 h are shown in Fig. 2. A defined concentration range of the enzymes (0.05-0.2 pg/pl each) was required to give A = T and G = C, as well as the expected values for the four major bases (see also Table 1). At low enzyme concentrations, digestion was not completed in 2 h. The drop in nucleotide count rates at high enzyme concentrations may conceivably be due to adsorption of nucleotides to protein occurring at such high protein:nucleotide ratios. When the concentration of micrococcal nuclease was varied, but DNA (0.1 pg/ ~1) and spleen phosphodiesterase (0.2 pg/ ~1) concentrations were kept constant, it was found that a range of 0.05 to 0.2 pg/pl of micrococcal nuclease gave satisfactory digestion. At higher and lower enzyme concentrations, recoveries of 32P-labeled nucleotides were again incomplete (data not shown). On the other hand, when the spleen phosphodiesterase concentration was increased to >0.2 rg/pl, such incomplete recoveries were not observed in reactions where DNA and micrococcal nuclease were kept at 0.1 and 0.2 pg/Fl, respectively (data not shown). The digestion of DNA (0.1 pg/pl) with micrococcal nuclease (0.2 pg/pl) and spleen phosphodiesterase (0.2 pg/pl) appeared complete in 15 min (Fig. 3). When the nonradioactive digest was analyzed directly by PEI-cellulose thin-layer chromatography, all the uv-absorbing material cochromatographed with authentic deoxynucleoside 3’monophosphates providing additional evidence that the digestion of DNA to mononucleotides was complete. On the basis of these results, we recommend the following digestion conditions: 0.05-0.3 pg/pl DNA, 0.1-0.2 pg/pl each for micrococcal nuclease and spleen phospho-
276
REDDY,
GUPTA,
AND TABLE
BASE COMPOSITION
ANALYSIS
OF VARIOUS
RANDERATH I DNAs
BY THE “P-LABELING
METHODS
Mel% Source
of DNA
G
C
A
T
m5C
Regenerating rat liver Our data“ Spectrophotometric analysis’
20.5 (o.20)b
28.7 (0.15)
20.6 (0.34)
0.72
(0.02)
29.7
21.1 (0.32)
27.9 (0.76)
21.5
0.77
(0.12)
28.7 (0.72)
Calf thymus Our data” Literature
21.8 (0.17) 19.5-23.9
27.3 (0.17) 27.1-30.3
20.8 (0.23) 20.0-22. I
1.07 (0.06) 1.1-1.9
28.9 (0.5 1) 26.6-31.2
22.7 (0.42)
28. I (0.30)
19.8 (0.41)
1.1 (0.05)
28.4 (0.46)
23.6 (0.28) 22.2-23.0
26. I (0.34) 27.8-29.0
19.8 (0.06) 20.4-2 1 .O
2.1 (0.06) 1.9
28.3 (0.37) 27.0-27.5
Salmon Our
range
testes data”
Herring sperm Our data” Literature range
(0.61)
” DNAs were digested, the products r2P-labeled, chromatographed, and base compositions under Materials and Methods. ’ Numbers in parentheses indicate standard deviations (IV = 4-6). ‘ DNA was digested with nuclease PI to deoxynucleoside 5’-monophosphates which were cellulose thin-layer chromatography, eluted, and assayed spectrophotometrically (Harris, K., unpublished).
diesterase, and incubation at 37°C for 30 to 120 min. Because of the dependence of the assay on the proper enzyme concentrations
I
I
4
60
120
Min
180
J
FIG. 3. DNA digestion with a 1:l mixture of micrococcal nuclease (0.2 @g/PI) and spleen phosphodiesterase (0.2 pg/pI) for different times. Regenerating rat liver DNA (0.1 @g/PI) was digested to deoxynucleoside 3’-monophosphates which were “P-labeled, chromatographed, and assayed as described under Materials and Methods. Notation defined as in Fig. 2.
analyzed
(0.26)
as described
then separated by PEIJ. S., and Randerath,
(Fig. 2) and the possible batch variations of the commercial enzyme preparations, it may be necessary to determine optimal conditions for fresh batches of the enzymes. Next we examined whether a minor nucleoside, such as m5C, in DNA could be determined by the 32P-labeling method. Treatment of the 32P-labeled digests with apyrase removes not only excess [ T-~*P]ATP but also several unidentified 32P-labeled contaminants in [T-~~P]ATP which would interfere with the detection and determination of minor DNA components. However, it did not remove radioactive background material remaining at the origin (Fig. 4) and several minor background compounds which became visible after prolonged autoradiography of the maps (data not shown); but overall, a higher degree of purification was achieved this way than by the periodate treatment referred to above. On some batches
“P-BASE
ANALYSIS
OF
DNA
277
in 0.8 M ammonium formate, pH 3.5, the spot was located by autoradiography, cut out, and treated in situ with nuclease P, to remove 3’-phosphate groups (29). When the product of this reaction was cochromatographed with authentic dpm5C by the contact transfer technique (29) in 0.1 N acetic acid (32) on a PEI-cellulose layer, all the radioactivity co-migrated with the uv-abFIG. 4. Two-dimensional separation of a “P-labeled sorbing material indicating that the original digest of DNA on a PEI-cellulose thin layer. Calf thyspot was dpm’Cp. mus DNA was digested to deoxynucleoside 3’-monoThe phosphorylation of deoxynucleoside phosphates which were 32P-labeled and chromato3’-monophosphates of the four major bases graphed according to the standard procedure described under Materials and Methods. The specific activity of and m5C was found to be complete in 10 to [T-‘~P]ATP was 10 Ci/mmol. After treatment with 15 min, and further addition of kinase did apyrase, “P-labeled digest (total radioactivity, 1.O &i; not alter the extent of labeling, as shown in 10 pmol of deoxynucleoside 3’,5’-bisphosphates) was Fig. 5. Chromatographic analyses of the rechromatographed in the first dimension (from bottom action mixtures (data not shown) indicated to top) with 0.8 M ammonium formate, pH 3.5, and in the second dimension (from left to right) with 0.3 M that most of the added [y-32P]ATP was still ammonium sulfate. Autoradiography was at -70°C for present at 180 min. In addition, our obser4 h. j2P, migrated ahead of the nucleotides in both divation that the radioactivity of the deoxymensions (not shown). nucleoside 3’,5’-bisphosphates remained constant over a period of several hours indicates of commercial PEI-cellulose sheets, the 32Pi the absence of 3’-phosphatase activity under spot traveled rather close to dpm’Cp and our incubation conditions. As also noted by Davies et al. (22), this is probably because dpCp and contributed background radioactivity to these compounds. In these cases, the 3’-phosphatase activity of T4 polynucleomitting the apyrase treatment improved the otide kinase is optimal at pH 6 (33) while our kinase reactions were performed at pH precision of m5C analysis (data not shown). In order to separate dpm’Cp from dpCp, we 9.0. Blanks calculated as described under chromatographed the 32P-labeled DNA hy- Materials and Methods were found to amount to 0.5 to 2% and 6 to 8% of the drolysate under different conditions from those given in Fig. 1; dpm5Cp was best resolved from dpCp by developing the PEIcellulose chromatograms for extended periods of time in 0.6 to 0.9 M ammonium formate, pH 3.5. Thus such a system was adopted for the first dimension of the separation, an example of which is shown in Fig. 4. As a comparison of Fig. 4 with Fig. Min 1 shows, this system gave a much better FIG. 5. Kinetics of phosphorylation of calf thymus overall resolution than the LiCl/formate DNA hydrolysate. DNA (0.1 pg/rl) was digested to combination. The 4 major deoxynucleoside deoxynucleoside 3’-monophosphates which were j2P-la3’,5’-bisphosphates were identified by co- beled, chromatographed, and assayed as described unchromatography with authentic markers. der Materials and Methods. At 180 min (arrow), more The spot above dpCp (Fig. 4) was shown polynucleotide kinase was added to 0.85 U//*1. Notation to be dpm5Cp as follows. After development defined as in Fig. 2.
REDDY,
278
GUPTA,
radioactivity of the major nucleotides m5C, respectively.
and
Base Analysis of Various DNA Samples by the Standard Procedure Table 1 shows the results of base composition analyses of various DNAs containing the 4 major bases and m5C by the standard procedure described under Materials and Methods. The data for calf thymus and herring sperm DNAs agreed well with the literature values (34) obtained by different methods. The values for 24-h regenerating rat liver DNA were close to values obtained by uv-spectrophotometric analysis (Table 1). They were also close to values reported by Vanyushin et al. (35) for normal rat liver DNA (G, 22.1; A, 28.5; C, 20.9; m5C, 0.94; and T, 27.6 mol%), except that our values for m5C are lower. Lapeyre and Becker (8) have also observed a decrease in DNA m5C content from 1.00 mol% for normal rat liver to 0.88 mol% for 2 1-h regenerating rat liver. Taken together, these results indicate that the standard procedure described in this paper leads to quantitative digestion of DNA to mixtures of deoxynucleoside 3’-monophosphates and to quantitative 32P-labeling of these compounds to [5’-32P]deoxynucleoside 3’,5’-bisphosphates; enabling one to determine the base composition of DNA by combining the digestion/32P-labeling scheme with thin-layer chromatography and counting of the labeled products. In view of the fact that the modified nucleoside, m5C, in DNA could be determined by this method, we have attempted to utilize the “P-labeling approach to detect other modified nucleosides, such as those formed by the binding of carcinogens to DNA. This work will be published elsewhere (36). ACKNOWLEDGMENTS We thank Dr. E. Randerath for Dr. J. S. Harris for a sample of generating rat liver, G. Gambhir assistance, and H. P. Agrawal for aration of the manuscript.
valuable discussions. DNA from 24-h refor expert technical help with the prep-
AND
RANDERATH
REFERENCES 1. Wyatt, G. R. (1955) in The Nucleic Acids (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, pp. 2433265, Academic Press, New York. 2. Randerath, K. (1965) Nature (London) 205, 908. 3. Rae, P. M. M., and Steele, R. E. (1978) BioSystems 10, 37-53. 4. Rubery, E. D., and Newton, A. A. (1973) Biochim. Biophys. Acta 324, 24-36. 5. Razin, A., and Sedat, J. ( 1977) Anal. Biochem. 77, 370-377. H. -J., and Zahn, R. K. (1973) Anal. 6. Breter, Biochem. 54, 346-352. 7. Yuki, H., Kawasaki, H., Imayuki, A., and Yajima, T. ( 1979) J. Chromatogr. 168, 489-494. 8. Lapeyre, J. -N., and Becker, F. F. (1979) Biochem. Biophys. Res. Commun. 87, 698-705. 9. Singer, J., Stellwagen, R. H., Roberts-Ems, J., and Riggs, A. D. (1977) .I. Biol. Chem. 252, 5509.. 5513. 10. Ehrlich, M., and Ehrlich, K. (1979) J. Chromatogr. sci. 17, 531-534. 11. Kuo, K. C., McCune, R. A., Gehrke, C. W.. Midgett, R., and Ehrlich, M. (1980) Nucleic Acids Rex 8, 4763 -4776. 12. Singer, J., Schnute, W. C. Jr.. Shively, J. E., Todd, C. W., and Riggs, A. D. (1979) Anal. Biochem. 94, 297.-301. 13. Deutsch, J., Razin, A., and Sedat, J. (I 976) Anal. Biochem. 72, 586-592. 14. Storl, H. J., Simon, H., and Barthelmes, H. ( 1979) Biochim. Biophys. Acta 564, 23-30. 15. Sano, H., Royer, H.-D., and Sager, R. ( 1980) Proc. Nat. Acad. Sci. (ISA 77, 3581-3585. 16. Hsu, 1. -C., Poirier, M. C., Yuspa, S. H., Grunberger, D., Weinstein, I. B., Yolken, R. H., and Harris,C. C. (198l)CancerRes. 41, 1091.-1095. 17. De Murcia, G., Lang, M.-C. E., Freund, A.-M., Fuchs, R. P. P., Daune, M. P., Sage, E., and Leng, M. ( 1979) Proc. Nat. Acad. Sri. USA 76, 6076-6080. 18. Randerath, K., Gupta, R. C., and Randerath, E. ( 1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 638-680, Academic Press, New York. 19. Simsek, M., Ziegenmeyer, J., Heckman, J., and RajBhandary, U. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1041-1045. 20. Agris, P. F.. Powers, T., Soli, D., and Ruddle, F. H. (1975) Cancer Biochem. Biophys. 1, 6977. 21. Maxam. A. M., and Gilbert, W. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 499-560, Academic Press, New York.
‘*P-BASE 22. 23.
24. 25. 26.
27.
28.
ANALYSIS
Davies, P. L., van de Sande, J. H., and Dixon, G. H. (1979) Anal. Eiochem. 93, 26-30. Randerath, K., Gupta, R. C., and Reddy, M. V. (198 1) J. Supramol. Struct. Cell. Biochem.. Suppl. 5, 173 (Abstract 463). Higgins, G. M., and Anderson, R. M. (1931) Arch. Pathol. 12, 186-202. Marmur, J. (1961) J. Mol. Biol. 3, 208-218. Randerath, K., and Randerath, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part A, pp. 323-347, Academic Press, New York. Sueoka, N., and Cheng, T. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part A, pp. 562-566, Academic Press, New York. Swanstrom, R., and Shank, P. R. (1978) Anal. Biochem. 86, 184- 192.
OF
29. 30. 31. 32. 33. 34.
35.
36.
DNA
279
Gupta, R. C., and Randerath, K. (1979) Nucleic Acids Rex 6, 3443-3458. Ohsaka, A., Mukai, J.-I., and Laskowski, M., Sr. (1964) J. Biol. Chem. 239, 3498-3504. Razzell, W. E., and Khorana, H. G. (1961) J. Biol. Chem. 236, 1144-l 149. Gupta, R. C., Randerath, E., and Randerath, K. (1976) Nucleic Acids Res. 3, 2915-2921. Cameron, V., and Uhlenbeck, 0. C. (1977) Biochemistry 16, 5 120-S 126. Shapiro, H. S. (1976) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., Vol. 2, pp. 241-283, CRC Press, Cleveland. Vanyushin, B. F., Mazin, A. L., Vasilyev, V. K., and Belozersky, A. N. (1973) Biochim. Biophys. Acta 299, 397-403. Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) Proc. Nat. Acad. Sci. USA, in press.
BIOCHEMISTRY
117, 271-279 (1981)
32P-Base
Analysis
of DNA’
M. VIJAYARAJ REDDY, RAMESH C. GUPTA, AND KURT RANDERATH Department
of Pharmacology,
Baylor
College
of Medicine,
Texas
Medical
Center.
Housron.
Texas
77030
Received March 31, 1981
A “P-labeling method for the base composition analysis of nonradioactive DNA was developed consisting of the digestion of DNA to deoxynucleoside 3’-monophosphates by incubation with a mixture of micr ococcal nuclease and spleen phosphodiesterase, transfer of ?label from [T-‘~P]ATF to the 5’-hydroxyl groups of the mononucleotides by T4 polynucleotide kinase, two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose of the resultant [ 5’-32P]deuxynucleoside 3’,5’-bisphosphates, autoradiography, and scintillation counting. The method was standardized to afford quantitative digestion of DNA to mononucleotides as well as to give quantitative incorporation of ?-label into the nucleotides in the DNA hydrolysate so as to make the method an accurate means for determining the base composition of eucaryotic DNA containing adenine, guanine, thymine, cytosine, and 5-methylcytosine.
The presence in DNA of bases other than adenine, guanine, thymine, and cytosine is a feature common to all organisms. Such modified bases may arise by enzymatic modification of DNA bases (i.e., 5-methylcytosine and N6-methyladenine) or by the action of chemicals (e.g., chemical carcinogens) on DNA. Several methods are available for the identification and quantitation of major and modified constituents of DNA. The earliest technique used for this purpose was paper chromatography (1). Paper and thin-layer chromatography (2) have also been used to analyze DNA that has been labeled in vivo with radioactive isotopes (3,4), an approach not well suited for the analysis of DNA from intact experimental animals and humans. More modern chromatographic techniques applied to this task have included gas chromatography (5), high-performance liquid chromatography (6-l l), and gas chromatography-mass spectrometry ( 12). Mass spectral ( 13) and immunological ( 14- 17)
methods have also been utilized to identify and to determine specific DNA components. Current methods for determining the base compositions and sequences of amounts of nonradioactive nucleic acids which are too small for conventional chromatography entail analysis by postlabeling techniques. Thus, for the base composition and sequence analysis of RNA (18-20) and for the sequence analysis of DNA (21), the nonradioactive nucleic acid is specifically broken down into smaller components which are then labeled with radioisotopes and are characterized by chromatographic or electrophoretic techniques. This approach has been particularly useful for the identification and the quantitation of modified constituents in RNA ( 18), but somewhat surprisingly has thus far not been applied to DNA base composition analysis. Davies et al. (22) recently reported experiments that demonstrate the feasibility of determining the composition of a mixture of the four major deoxynucleoside 3’-monophosphates by a 32P-postlabeling technique, but these authors have not applied this technique to the analysis of DNA.
’ Supported by USPHS grant CA-25590.
271
0003-2697/81/160271-09$02.00/O Copyright 0 198 I by Academic Press, Inc. All rights of reproduction in any form reserved.
REDDY,
272
GUPTA,
This article details a 32P-postlabeling method for base composition analysis of DNA which consists of the enzymatic digestion of DNA to deoxynucleoside 3’monophosphates, conversion of these compounds to [ 5’-32PJdeoxynucleoside 3’,5’bisphosphates by the T4 polynucleotide kinase-catalyzed phosphorylation reaction, separation of the 32P-labeled nucleotides by anion-exchange thin-layer chromatography on PEI-cellulose,’ and quantitation of these nucleotides by scintillation counting. This work has been presented in preliminary form (23). MATERIALS
AND METHODS
Materials
2’-Deoxynucleoside 3’,5’-bisphosphates (PL Biochemicals), 5-methyl-2’-deoxycytidine 5’-phosphate (Sigma Chemical Co. ), NJVbis(2-hydroxyethyl)glycine (Bicine) (Calbiochem), [Y-~~PJATP (Amersham), T4 polynucleotide kinase (P-L Biochemicals), and potato apyrase (grade I, Sigma Chemical Co.) were used without further purification. The hexanucleotide, dATGCAT (Collaborative Research, Inc.), was purified by PEI-cellulose thin-layer chromatography ( 18) before use. Micrococcal nuclease (grade VI, Sigma Chemical Co., 0.21 U/rg) and spleen phosphodiesterase (BoehringerMannheim, 0.002 U/pg) were dialyzed against water at 4°C for 15 h. Female Sprague-Dawley rats weighing 150- 185 g were partially hepatectomized according to the method of Higgins and Anderson (24) and liver DNA was isolated after 24 h by a modification of the procedure of Marmur (25). This and other DNA preparations (Sigma Chemical Co.) were treated with 0.3 N KOH at 37°C for 15 h, neutralized with 1 N HCl, precipitated with alcohol, and dialyzed against water at 4°C for 15 h to remove ribonucleotides. PEI-cellulose sheets (20 x 20 cm) without indicator (Brinkmann 2 Abbreviations Smethylcytosine.
used:
PEI,
polyethyleneimine;
mSC,
AND
RANDERATH
Instruments, Inc.) were prewashed as described (26) prior to use. XAR-5 films (Eastman-Kodak Co.) and Lighting Plus intensifying screens (DuPont) were used for autoradiography. Concentrations of dATGCAT and DNA were estimated by uv absorption at 260 nm at neutral pH using units/mg for dATGCAT (from 29.9 A260nm data sheet of the supplier) and 20 &Onm units/mg for native DNA (27). Model Experiments 32P-labeling of model nucleotide mixture.
An equimolar mixture of dAp, dGp, dCp, and dTp (total concentration, 6 PM), [y32P]ATP (60 PM, 10 Ci/mmol), and T4 polynucleotide kinase (0.25 U/PI) in 10 ~1 of a solution containing 40 mM Bicine-NaOH, 10 mM MgC12, 10 mM dithiothreitol, and 0.1 mM spermidine, pH 9.0, was incubated at 37°C for 60 min. Control reactions were conducted without the addition of the nucleotides, but were further processed like samples containing the nucleotides. A 2-~1 aliquot of the 32P-labeled reaction mixture was added to 8 ~1 of a solution containing 30 mrvr sodium periodate, 50 mM sodium acetate, and 12 mM EDTA, pH 5.0, and incubated at 22°C for 10 min. After the addition of 2 ~1 of a solution containing 4 hg each of dpAp, dpGp, dpCp, and dpTp, the mixture was applied to a PEI-cellulose sheet (10 X 20 cm) at a point 2.5 cm from both the left-hand and bottom edges. Development in the first (short) dimension was with water to the origin, followed by 1.5 M LiCl to the top (-7.5 cm from the origin). The sheet was dried, soaked in 500 ml of methanol with gentle shaking for 10 min, and dried. For the second (long) dimension, chromatography was with 1.75 M ammonium formate, pH 3.5,3 to 2 cm on a Whatman No. 1 wick attached to the top of the sheet (- 17.5 cm from the origin). Individual ’ This prepared hydroxide.
and other ammonium by titration of formic
formate solutions were acid with ammonium
‘*P-BASE
ANALYSIS
nucleotide spots were located by screen-intensified autoradiography (28), cut out, and their radioactivity was determined by Cerenkov counting. Equivalent areas were cut from control chromatograms and were counted. These blanks were subtracted from the sample values. Base ratios were calculated directly from the corrected values and were expressed as percentages of the total. Studies with dATGCAT. The compound (1 pg) was hydrolyzed to deoxynucleoside 3’-monophosphates with 2 rg of spleen phosphodiesterase in 10 ~1 of 20 mM sodium succinate, pH 6.0, at 37°C for 2 h. Control reactions were conducted without the addition of dATGCAT and were further processed like samples containing nucleotides. The nucleotides in the digest were 32P-labeled, chromatographed after treatment with periodate, and assayed as described above. Standardization of Conditions for DNA Base Analysis DNA digestion. In these experiments, which were designed to optimize the digestion conditions, either enzyme:substrate ratios or digestion times were varied. In the first set of experiments, 1 pg of DNA was digested with varying amounts of a 1: 1 mixture of micrococcal nuclease and spleen phosphodiesterase at 37°C for 2 h in 10 ~1 of a solution containing 20 mrvt sodium succinate and 8 mM CaCl,, pH 6.0. In the second set of experiments, 1 pg of DNA was digested at 37°C for different time intervals with 2 pg each of micrococcal nuclease and spleen phosphodiesterase in 10 ~1 of the same buffer. The nucleotides in the digests were 32P-labeled and chromatographed, and their radioactivity was determined as described above. 32P-labeling of nucleotides in DNA digests. To standardize the labeling conditions,
DNA was digested to deoxynucleoside 3’monophosphates which were 32P-labeled and chromatographed as described in the following section except that the 32P-labeling re-
OF
DNA
273
action was conducted for various intervals of time, and additional T4 polynucleotide kinase (final, 0.85 U/pi) was added at 180 min. Standard Procedure for Quantitative Analysis of DNA
Base
Digestion and “P-labeling. For quantitative digestion of DNA to deoxynucleoside 3’-monophosphates, 1 pg of DNA (- 3 nmol of DNA-P) was incubated at 37°C for 2 h with 2 pg each of micrococcal nuclease and spleen phosphodiesterase in 10 ~1 of a solution containing 20 mrvr sodium succinate and 8 mM CaC12, pH 6.0. Control reactions were conducted without DNA and were further processed like the samples containing nucleotides. The digest was diluted IO-fold and a 2-J aliquot containing -60 pmol of deoxynucleoside 3’-monophosphates was mixed with a solution of [T-~*P]ATP (60 PM, 1O-20 Ci/mmol) and T4 polynucleotide kinase (0.25 U/pi) in 10 ~1 of a solution containing 40 mM Bicine-NaOH, 10 mM MgC12, 10 mM dithiothreitol, and 0.1 mM spermidine, pH 9.0. Incubation was at 37°C for 1 h. Chromatography and quantitative analysis. The 32P-labeled mixture (10 ~1) was
incubated with 2.5 cclof a solution of potato apyrase (5 mU/pl) at 37°C for 1 h to convert excess [ T-~*P]ATP and 32P-labeled impurities (see Results and Discussion) to 32Pi before chromatography. Aliquots (2 ~1) of the reaction mixture were applied to each of 4 to 6 PEI-cellulose sheets (20 X 20 cm) which had been soaked in 500 ml of 0.1 M ammonium formate, pH 3.5, for 30 min and dried, first in a current of cool air, then warm air (5 min each). The sheets were developed overnight, at least 15 h, in 0.8 M ammonium formate, pH 3.5, to about 25 cm on a Whatman No. 1 wick which had been attached to the top of the sheet by stapling and then had been folded. Development was then continued for 2 to 3 h in 4 M ammonium formate, pH 3.5, without intermediate drying.
REDDY,
214
GUPTA,
The sheets were dried as above, soaked in 250 ml of methanol for 10 min, and dried briefly in cool air. For the second dimension, chromatography was with water to the origin, followed by 0.3 M ammonium sulfate to 4 to 5 cm on a Whatman No. 1 wick attached to the top of the sheet. Individual nucleotide spots were located by autoradiography, cut from the chromatogram, and their radioactivity was determined by Cerenkov counting. Base ratios were calculated, after subtracting blank values, from the 4 to 6 replicate analyses as described above.
AND
RANDERATH
j2P-Labeling of model nucleotide mixture. Davies et al. (22) have reported that
FIG. 1. Two-dimensional separation of a “P-labeled digest of dATGCAT on a PEI-cellulose thin-layer sheet. The deoxynucleoside 3’-monophosphates obtained by enzymatic digestion of the hexanucleotide were “P-labeled and chromatographed as described under Materials and Methods. The specific activity of [y-‘*P]ATP was 10 Ci/mmol. After NaI04 treatment, the ‘*P-labeled digest of dATGCAT (total radioactivity, 1.3 &i; 12 pmol of deoxynucleoside 3’,.5’-bisphosphates) was applied to the chromatogram. Development in the first dimension (from bottom to top) was with 1.5 M LiCI, and in the second dimension (from left to right) with 1.75 M ammonium formate, pH 3.5. Autoradiography was at -70°C for 11 h.
a mixture of dAp, dGp, dCp, and dTp (total concentration, 6 PM) could be quantitatively phosphorylated by incubation with 60 I.LM [ Y-~*P]ATP and 0.06 U/p1 of T4 polynucleotide kinase at pH 9.2 and 37°C for 30 min as assayed by DEAE-paper electrophoresis and counting. Using the conditions described under Materials and Methods, we also found the phosphorylation of deoxynucleoside 3’monophosphates to proceed to completion. These conditions, which are slightly different from those given by Davies et al. (22), have been adapted to give optimal phosphorylation rates with a commercial preparation of T4 polynucleotide kinase. In addition, our procedure specifies ion-exchange thin-layer chromatography to separate the 32P-labeled nucleotides (see Fig. 1) because this technique affords better separations of modified nucleotides than does DEAE-paper electrophoresis (29). Before chromatography, the mixture of 32P-labeled nucleotides was treated with sodium periodate to convert excess [Y-~*P]ATP to the dialdehyde, most of which was retained at the origin by binding to the amino groups of polyethyleneimine during subsequent chromatography. Studies with dATGCAT. Davies et al. (22) have shown that the base composition
of a ribooligonucleotide could be determined by enzymatic digestion to ribonucleoside 3’monophosphates and enzymatic 32P-labeling. These authors did not report an extension of this approach to deoxyoligonucleotides or DNA. We have chosen dATGCAT as a model compound to test the accuracy and precision of the “P-labeling method for the determination of the base composition of a deoxyoligonucleotide. This oligonucleotide was digested to deoxynucleoside 3’monophosphates and the products of the 32Plabeling reaction were chromatographed after treatment of the reaction mixture with sodium periodate as described under Materials and Methods. The resolution of ‘*P-labeled deoxynucleoside 3’,5’-bisphosphates is shown in Fig. 1. Individual ‘*P-labeled compounds were identified by cochromatography with authentic markers, and their radioactivity was determined. In addition to 32P-labeled nucleotides and 32Pi,several other spots contributed by [T-~*P]ATP were visible after prolonged exposure (Fig. 1). The bulk of [T-~*P]ATP was retained at the origin. The expected base ratio of A:G:C:T for this compound is 2:l: 1: 1 since thymidine derived
RESULTS AND DISCUSSION
Model Experiments
“P-BASE
ANALYSIS
from the 3’-terminus by spleen phosphodiesterase digestion is not a substrate of the polynucleotide kinase reaction. The average base ratio calculated from 8 determinations was 2.02 (+0.057):1.01 (f0.051):0.94 ( IL 0.040): 1.04 ( It_0.033). Standardization of conditions for DNA analysis. Subsequently, conditions were
standardized for the digestion and base composition analysis of DNA containing the four major bases and m5C. The accuracy of these determinations is dependent on obtaining quantitative hydrolysis of DNA and quantitative labeling of the digestion products. We have chosen a mixture of micrococcal nuclease and spleen phosphodiesterase for the digestion of DNA because the former enzyme digests DNA to a mixture of deoxynucleoside 3’-monophosphates and dinucleotides carrying S-hydroxyls and 3’phosphates (30) while the latter enzyme digests oligonucleotides carrying 5’-hydroxyls to nucleoside 3’-monophosphates (3 1). Thus, a combination of the two enzymes was expected to afford quantitative hydrolysis of DNA to the deoxynucleoside 3’-monophosphate level.
16
-
ro 12 k? x6E . Et4 I
T C A G I 0.01
I
11111110 0.1
0.03
1 * 111.111 0.3 I
1
I
2
Micrococcal Nuclease + Spleen Phosphodiesterase (pg/ul each) FIG. 2. DNA digestion with varying amounts of a 1:l mixture of micrococcal nuclease and spleen phosphodiesterase. Regenerating rat liver DNA (0.1 pg/pl) was digested to deoxynucleoside 3’-monophosphates which were “P-labeled, chromatographed, and assayed as described under Materials and Methods. Chromatography in LiCl/ammonium formate solvents (see Materials and Methods) did not separate dpm’Cp and dpCp; thus C denotes the sum of dpCp and dpm’Cp. T denotes dpTp; G, dpGp; and A, dpAp.
OF
DNA
275
DNA digestion was studied by varying enzyme and DNA concentrations. The digests were subjected to quantitative ‘*P-labeling and chromatographic analyses. The results obtained by digesting DNA (0. I pg/ ~1) from regenerating rat liver with varying amounts of micrococcal nuclease and spleen phosphodiesterase at 37°C for 2 h are shown in Fig. 2. A defined concentration range of the enzymes (0.05-0.2 pg/pl each) was required to give A = T and G = C, as well as the expected values for the four major bases (see also Table 1). At low enzyme concentrations, digestion was not completed in 2 h. The drop in nucleotide count rates at high enzyme concentrations may conceivably be due to adsorption of nucleotides to protein occurring at such high protein:nucleotide ratios. When the concentration of micrococcal nuclease was varied, but DNA (0.1 pg/ ~1) and spleen phosphodiesterase (0.2 pg/ ~1) concentrations were kept constant, it was found that a range of 0.05 to 0.2 pg/pl of micrococcal nuclease gave satisfactory digestion. At higher and lower enzyme concentrations, recoveries of 32P-labeled nucleotides were again incomplete (data not shown). On the other hand, when the spleen phosphodiesterase concentration was increased to >0.2 rg/pl, such incomplete recoveries were not observed in reactions where DNA and micrococcal nuclease were kept at 0.1 and 0.2 pg/Fl, respectively (data not shown). The digestion of DNA (0.1 pg/pl) with micrococcal nuclease (0.2 pg/pl) and spleen phosphodiesterase (0.2 pg/pl) appeared complete in 15 min (Fig. 3). When the nonradioactive digest was analyzed directly by PEI-cellulose thin-layer chromatography, all the uv-absorbing material cochromatographed with authentic deoxynucleoside 3’monophosphates providing additional evidence that the digestion of DNA to mononucleotides was complete. On the basis of these results, we recommend the following digestion conditions: 0.05-0.3 pg/pl DNA, 0.1-0.2 pg/pl each for micrococcal nuclease and spleen phospho-
276
REDDY,
GUPTA,
AND TABLE
BASE COMPOSITION
ANALYSIS
OF VARIOUS
RANDERATH I DNAs
BY THE “P-LABELING
METHODS
Mel% Source
of DNA
G
C
A
T
m5C
Regenerating rat liver Our data“ Spectrophotometric analysis’
20.5 (o.20)b
28.7 (0.15)
20.6 (0.34)
0.72
(0.02)
29.7
21.1 (0.32)
27.9 (0.76)
21.5
0.77
(0.12)
28.7 (0.72)
Calf thymus Our data” Literature
21.8 (0.17) 19.5-23.9
27.3 (0.17) 27.1-30.3
20.8 (0.23) 20.0-22. I
1.07 (0.06) 1.1-1.9
28.9 (0.5 1) 26.6-31.2
22.7 (0.42)
28. I (0.30)
19.8 (0.41)
1.1 (0.05)
28.4 (0.46)
23.6 (0.28) 22.2-23.0
26. I (0.34) 27.8-29.0
19.8 (0.06) 20.4-2 1 .O
2.1 (0.06) 1.9
28.3 (0.37) 27.0-27.5
Salmon Our
range
testes data”
Herring sperm Our data” Literature range
(0.61)
” DNAs were digested, the products r2P-labeled, chromatographed, and base compositions under Materials and Methods. ’ Numbers in parentheses indicate standard deviations (IV = 4-6). ‘ DNA was digested with nuclease PI to deoxynucleoside 5’-monophosphates which were cellulose thin-layer chromatography, eluted, and assayed spectrophotometrically (Harris, K., unpublished).
diesterase, and incubation at 37°C for 30 to 120 min. Because of the dependence of the assay on the proper enzyme concentrations
I
I
4
60
120
Min
180
J
FIG. 3. DNA digestion with a 1:l mixture of micrococcal nuclease (0.2 @g/PI) and spleen phosphodiesterase (0.2 pg/pI) for different times. Regenerating rat liver DNA (0.1 @g/PI) was digested to deoxynucleoside 3’-monophosphates which were “P-labeled, chromatographed, and assayed as described under Materials and Methods. Notation defined as in Fig. 2.
analyzed
(0.26)
as described
then separated by PEIJ. S., and Randerath,
(Fig. 2) and the possible batch variations of the commercial enzyme preparations, it may be necessary to determine optimal conditions for fresh batches of the enzymes. Next we examined whether a minor nucleoside, such as m5C, in DNA could be determined by the 32P-labeling method. Treatment of the 32P-labeled digests with apyrase removes not only excess [ T-~*P]ATP but also several unidentified 32P-labeled contaminants in [T-~~P]ATP which would interfere with the detection and determination of minor DNA components. However, it did not remove radioactive background material remaining at the origin (Fig. 4) and several minor background compounds which became visible after prolonged autoradiography of the maps (data not shown); but overall, a higher degree of purification was achieved this way than by the periodate treatment referred to above. On some batches
“P-BASE
ANALYSIS
OF
DNA
277
in 0.8 M ammonium formate, pH 3.5, the spot was located by autoradiography, cut out, and treated in situ with nuclease P, to remove 3’-phosphate groups (29). When the product of this reaction was cochromatographed with authentic dpm5C by the contact transfer technique (29) in 0.1 N acetic acid (32) on a PEI-cellulose layer, all the radioactivity co-migrated with the uv-abFIG. 4. Two-dimensional separation of a “P-labeled sorbing material indicating that the original digest of DNA on a PEI-cellulose thin layer. Calf thyspot was dpm’Cp. mus DNA was digested to deoxynucleoside 3’-monoThe phosphorylation of deoxynucleoside phosphates which were 32P-labeled and chromato3’-monophosphates of the four major bases graphed according to the standard procedure described under Materials and Methods. The specific activity of and m5C was found to be complete in 10 to [T-‘~P]ATP was 10 Ci/mmol. After treatment with 15 min, and further addition of kinase did apyrase, “P-labeled digest (total radioactivity, 1.O &i; not alter the extent of labeling, as shown in 10 pmol of deoxynucleoside 3’,5’-bisphosphates) was Fig. 5. Chromatographic analyses of the rechromatographed in the first dimension (from bottom action mixtures (data not shown) indicated to top) with 0.8 M ammonium formate, pH 3.5, and in the second dimension (from left to right) with 0.3 M that most of the added [y-32P]ATP was still ammonium sulfate. Autoradiography was at -70°C for present at 180 min. In addition, our obser4 h. j2P, migrated ahead of the nucleotides in both divation that the radioactivity of the deoxymensions (not shown). nucleoside 3’,5’-bisphosphates remained constant over a period of several hours indicates of commercial PEI-cellulose sheets, the 32Pi the absence of 3’-phosphatase activity under spot traveled rather close to dpm’Cp and our incubation conditions. As also noted by Davies et al. (22), this is probably because dpCp and contributed background radioactivity to these compounds. In these cases, the 3’-phosphatase activity of T4 polynucleomitting the apyrase treatment improved the otide kinase is optimal at pH 6 (33) while our kinase reactions were performed at pH precision of m5C analysis (data not shown). In order to separate dpm’Cp from dpCp, we 9.0. Blanks calculated as described under chromatographed the 32P-labeled DNA hy- Materials and Methods were found to amount to 0.5 to 2% and 6 to 8% of the drolysate under different conditions from those given in Fig. 1; dpm5Cp was best resolved from dpCp by developing the PEIcellulose chromatograms for extended periods of time in 0.6 to 0.9 M ammonium formate, pH 3.5. Thus such a system was adopted for the first dimension of the separation, an example of which is shown in Fig. 4. As a comparison of Fig. 4 with Fig. Min 1 shows, this system gave a much better FIG. 5. Kinetics of phosphorylation of calf thymus overall resolution than the LiCl/formate DNA hydrolysate. DNA (0.1 pg/rl) was digested to combination. The 4 major deoxynucleoside deoxynucleoside 3’-monophosphates which were j2P-la3’,5’-bisphosphates were identified by co- beled, chromatographed, and assayed as described unchromatography with authentic markers. der Materials and Methods. At 180 min (arrow), more The spot above dpCp (Fig. 4) was shown polynucleotide kinase was added to 0.85 U//*1. Notation to be dpm5Cp as follows. After development defined as in Fig. 2.
REDDY,
278
GUPTA,
radioactivity of the major nucleotides m5C, respectively.
and
Base Analysis of Various DNA Samples by the Standard Procedure Table 1 shows the results of base composition analyses of various DNAs containing the 4 major bases and m5C by the standard procedure described under Materials and Methods. The data for calf thymus and herring sperm DNAs agreed well with the literature values (34) obtained by different methods. The values for 24-h regenerating rat liver DNA were close to values obtained by uv-spectrophotometric analysis (Table 1). They were also close to values reported by Vanyushin et al. (35) for normal rat liver DNA (G, 22.1; A, 28.5; C, 20.9; m5C, 0.94; and T, 27.6 mol%), except that our values for m5C are lower. Lapeyre and Becker (8) have also observed a decrease in DNA m5C content from 1.00 mol% for normal rat liver to 0.88 mol% for 2 1-h regenerating rat liver. Taken together, these results indicate that the standard procedure described in this paper leads to quantitative digestion of DNA to mixtures of deoxynucleoside 3’-monophosphates and to quantitative 32P-labeling of these compounds to [5’-32P]deoxynucleoside 3’,5’-bisphosphates; enabling one to determine the base composition of DNA by combining the digestion/32P-labeling scheme with thin-layer chromatography and counting of the labeled products. In view of the fact that the modified nucleoside, m5C, in DNA could be determined by this method, we have attempted to utilize the “P-labeling approach to detect other modified nucleosides, such as those formed by the binding of carcinogens to DNA. This work will be published elsewhere (36). ACKNOWLEDGMENTS We thank Dr. E. Randerath for Dr. J. S. Harris for a sample of generating rat liver, G. Gambhir assistance, and H. P. Agrawal for aration of the manuscript.
valuable discussions. DNA from 24-h refor expert technical help with the prep-
AND
RANDERATH
REFERENCES 1. Wyatt, G. R. (1955) in The Nucleic Acids (Chargaff, E., and Davidson, J. N., eds.), Vol. 1, pp. 2433265, Academic Press, New York. 2. Randerath, K. (1965) Nature (London) 205, 908. 3. Rae, P. M. M., and Steele, R. E. (1978) BioSystems 10, 37-53. 4. Rubery, E. D., and Newton, A. A. (1973) Biochim. Biophys. Acta 324, 24-36. 5. Razin, A., and Sedat, J. ( 1977) Anal. Biochem. 77, 370-377. H. -J., and Zahn, R. K. (1973) Anal. 6. Breter, Biochem. 54, 346-352. 7. Yuki, H., Kawasaki, H., Imayuki, A., and Yajima, T. ( 1979) J. Chromatogr. 168, 489-494. 8. Lapeyre, J. -N., and Becker, F. F. (1979) Biochem. Biophys. Res. Commun. 87, 698-705. 9. Singer, J., Stellwagen, R. H., Roberts-Ems, J., and Riggs, A. D. (1977) .I. Biol. Chem. 252, 5509.. 5513. 10. Ehrlich, M., and Ehrlich, K. (1979) J. Chromatogr. sci. 17, 531-534. 11. Kuo, K. C., McCune, R. A., Gehrke, C. W.. Midgett, R., and Ehrlich, M. (1980) Nucleic Acids Rex 8, 4763 -4776. 12. Singer, J., Schnute, W. C. Jr.. Shively, J. E., Todd, C. W., and Riggs, A. D. (1979) Anal. Biochem. 94, 297.-301. 13. Deutsch, J., Razin, A., and Sedat, J. (I 976) Anal. Biochem. 72, 586-592. 14. Storl, H. J., Simon, H., and Barthelmes, H. ( 1979) Biochim. Biophys. Acta 564, 23-30. 15. Sano, H., Royer, H.-D., and Sager, R. ( 1980) Proc. Nat. Acad. Sci. (ISA 77, 3581-3585. 16. Hsu, 1. -C., Poirier, M. C., Yuspa, S. H., Grunberger, D., Weinstein, I. B., Yolken, R. H., and Harris,C. C. (198l)CancerRes. 41, 1091.-1095. 17. De Murcia, G., Lang, M.-C. E., Freund, A.-M., Fuchs, R. P. P., Daune, M. P., Sage, E., and Leng, M. ( 1979) Proc. Nat. Acad. Sri. USA 76, 6076-6080. 18. Randerath, K., Gupta, R. C., and Randerath, E. ( 1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 638-680, Academic Press, New York. 19. Simsek, M., Ziegenmeyer, J., Heckman, J., and RajBhandary, U. L. (1973) Proc. Nat. Acad. Sci. USA 70, 1041-1045. 20. Agris, P. F.. Powers, T., Soli, D., and Ruddle, F. H. (1975) Cancer Biochem. Biophys. 1, 6977. 21. Maxam. A. M., and Gilbert, W. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 499-560, Academic Press, New York.
‘*P-BASE 22. 23.
24. 25. 26.
27.
28.
ANALYSIS
Davies, P. L., van de Sande, J. H., and Dixon, G. H. (1979) Anal. Eiochem. 93, 26-30. Randerath, K., Gupta, R. C., and Reddy, M. V. (198 1) J. Supramol. Struct. Cell. Biochem.. Suppl. 5, 173 (Abstract 463). Higgins, G. M., and Anderson, R. M. (1931) Arch. Pathol. 12, 186-202. Marmur, J. (1961) J. Mol. Biol. 3, 208-218. Randerath, K., and Randerath, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part A, pp. 323-347, Academic Press, New York. Sueoka, N., and Cheng, T. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part A, pp. 562-566, Academic Press, New York. Swanstrom, R., and Shank, P. R. (1978) Anal. Biochem. 86, 184- 192.
OF
29. 30. 31. 32. 33. 34.
35.
36.
DNA
279
Gupta, R. C., and Randerath, K. (1979) Nucleic Acids Rex 6, 3443-3458. Ohsaka, A., Mukai, J.-I., and Laskowski, M., Sr. (1964) J. Biol. Chem. 239, 3498-3504. Razzell, W. E., and Khorana, H. G. (1961) J. Biol. Chem. 236, 1144-l 149. Gupta, R. C., Randerath, E., and Randerath, K. (1976) Nucleic Acids Res. 3, 2915-2921. Cameron, V., and Uhlenbeck, 0. C. (1977) Biochemistry 16, 5 120-S 126. Shapiro, H. S. (1976) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., Vol. 2, pp. 241-283, CRC Press, Cleveland. Vanyushin, B. F., Mazin, A. L., Vasilyev, V. K., and Belozersky, A. N. (1973) Biochim. Biophys. Acta 299, 397-403. Randerath, K., Reddy, M. V., and Gupta, R. C. (1981) Proc. Nat. Acad. Sci. USA, in press.