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Enzymatic Synthesis of Deoxyribonucleic Acids with Repeating Sequences. A New Repeating Trinucleotide Deoxyribonucleic Acid, d( T-C-C),*d(G-G-A),t A. Richard Morgan,* Marion B. Coulter, Wayne F. Flintoff, and Verner H. Paetkau
ABSTRACT: Synthetic DNAs with repeating nucleotide sequences have been valuable as models for more complex DNAs. The enzymatic copying of synthetic DNAs poses several problems including (a) production of covalent links between complementary strands (clc-DNA) and (b) contamination of the product with other polymers, such as d(A-T),.d(A-T), and dG,.dC,. This paper details (i) the optimum conditions for
the enzymatic copying of synthetic DNAs, (ii) a simple fluorescence assay for the extent of synthesis and the presence of clc-DNA, (iii) a quick isolation procedure, (iv) conditions necessary for producing DNAs without clc strands, (v) a very sensitive assay for estimating contaminating d(A-T), . d(A-T)*, and (vi) the synthesis of a new polymer d(T-C-C),. d(G-G-A), and its physical properties.
S
Materials and Methods.
t From the Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada. Receiued Februarji 20, 1973. This work was supported by the Medical Research Council of Canada, Grants MA3758 and MA3764. Abbreviation used is: clc-DNA, DNA with covalently linked, complementary sequences.
Chemicals. d(C-C-T)4 and d(G-G-A)3 were chemically synthesized in Dr. H. G. Khorana’s laboratory (Narang et al., 1967). The other DNA templates with repeating sequences have already been described (Wells et al., 1967). Nucleases were from Worthington Biochemical Corp., ethidium bromide from Sigma Chemical Co., Agarose 5m from Bio-Rad Labs, CsCl was from Pierce Chemical Co. and Schwarz/Mann, and Cs2S04 from Schwarz/Mann. Radioactive (I4C and 3H) nucleoside and deoxynucleoside triphosphate were obtained from Schwarz/Mann, unlabeled nucleotides from P-L Biochemicals and Raylo Co., and [a3*-P]dATPand -dCTP from International Chemical and Nuclear Corp. E. coli B cells, grown to “14 log phase in minimal medium, were purchased frozen from the Grain Processing Co., Muscatine, Iowa. DNA polymerase I from E. coli was prepared by the method of Jovin et al. (1969), and RNA polymerase by a modification (Paetkau and Coy, 1972) of the procedure of Chamberlin and Berg (1962). A by-product of RNA polymerase preparation was fraction DIII. Material extracted from the protamine pellet with 0.01 M Tris-HC1 (pH 8.0)0.1 M MgC12-0.1 mM EDTA-10 mM /3-mercaptoethanol was fractionated with ammonium sulfate and the fraction precipitating between 45 and 65% saturation redissolved in 20 mM potassium phosphate (pH 7.4) as DIII (protein concentration 1.8 mgiml). DIII was added to prevent the accumulation of clc strands during the DNA polymerase reaction. Fraction IV DNA polymerase (Richardson et al., 1964) was also effective in preventing clc sequences (Paetkau, 1969) and was used in some experiments instead of DIII. The factor in DIII blocking clc accumulation is a heat-stable protein other than endonuclease I (Coulter et al., 1974). DIII was heated (97” for 10 min) to remove most of the nuclease activity, and tRNA was added to the DNA polymerase reactions to block residual endonuclease I activity, as indicated in the experiments. Pancreatic DNase was added in some experiments to stimulate synthesis, as indicated. Assays. The DNA polymerase reaction mixtures contained : 50 mM potassium phosphate (pH 7.5), 7-15 mM MgCh depending on the triphosphate concentration, deoxynucleoside triphosphate 0.8-8 mM total in a ratio corresponding to the base ratio of the template, 20 pgjml of DNA polymerase I
ynthetic DNAs with repeating nucleotide sequences were originally chemically synthesized and enzymatically copied with DNA polymerase I of Escherichia coli in the laboratory of Dr. H. G. Khorana (Byrd et ai., 1965; Wells et al., 1967). They have been used in many types of studies both biological and physicochemical, for example, the elucidation of the genetic code (Khorana et al., 1966) and the specificity of three-stranded helices (Morgan and Wells, 1968). Difficulties had arisen in synthesizing DNAs with high GC content (R. D. Wells, unpublished data). Under conditions successfully used for copying synthetic polymers with DNA polymerase, the expected product d(T-C-C), * d(G-G-A), was not formed from the mixture of oligomers d(C-C-T)4 and d(G-G-A)3. By altering the conditions of synthesis it is now possible to make d(T-C-C), d(G-G-A), and also to improve considerably on the net-fold replication of other synthetic DNAs. We routinely follow the synthesis of DNA by a very sensitive fluorescence assay which depends on the intercalation of ethidium bromide between the bases in duplex structures specifically (Le Pecq and Paoletti, 1966, 1967; Morgan and Paetkau, 1972). This assay is not only sensitive, but an immediate reading is obtained allowing calculation of the amount of DNA synthesized. If degradative nuclease action is beginning to overtake the synthetic reaction, isolation of DNA can be initiated at once. The fluorescence assay also allows a determination of the amount of clcl-DNA present, and since d(A-T),‘d(A-T), is a frequent contaminant of synthetic reactions (Burd and Wells, 1970) it can usually be estimated by the fluorescence assay. An alternative assay for d(A-T),.d(A-T),, which will detect at little as 0.01 contamination, depends on transcription using ATP and UTP as the only rNTP substrates, at relatively high KC1 concentrations (0.15-0.2 M). We have developed a simple gel filtration procedure for isolating DNAs in any desired buffer within 10-20 min of terminating the reaction.
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SYNTHESIS
fraction 7 (Jovin et al., 1969), either fraction 4 or DIII, and the DNA template, as indicated. The RNA polymerase reaction mixtures contained: 40 mM Tris-HC1 (pH 8), 5 mM MgC12, 0.15 M KC1, 0.2 mM dithiothreitol, 0.5 mM of each required rNTP, 0.15 mg/ml of RNA polymerase, and the template concentration as indicated. [14C]UTP was 1580 cpm/nmol and RNA synthesis was measured by acid insolubility as previously described (Nishimura et al., 1964). The synthesis of DNA was followed either by the incorporation of radioactive deoxynucleoside triphosphate into acid-insoluble material (Nishimura et al., 1964) or by the enhanced fluorescence of ethidium bromide. The standard ethidium bromide solution for assaying DNA contained 2 mM Tris-HC1 (pH 8), 0.2 mM EDTA, and 0.5 pg/ml of ethidium bromide. The excitation wavelength was 525 nm and the emission was 600 nm, using the Turner spectrofluorometer, Model 430; 2 ml of standard ethidium bromide solution was added to 1-cm2 cuvets (Spectrosil) as a blank, and a sample of DNA (up to 20 p1 volume) was added to a second cuvet with 2 ml of standard ethidium bromide solution. Routine precautions included calibrating the instrument from time to time with a fluorescent standard, as the intensity of the xenon lamp sometimes fluctuated. This fluorescence assay responded linearly to DNA concentrations up to 1 pg/ml (Morgan and Paetkau, 1972). None of the components of the DNA polymerase reaction mixture interfered with the fluorescence assay under our conditions. All measurements were at 25 '. CsCl Density Gradient Centrijugation. Analytical CsCl density gradient centrifugation was carried out on the Beckman Model E ultracentrifuge with uv optics. Centrifugaton was at 48,000 rpm for at least 20 hr at 25". The density of the solution was determined by the refractive index on an Abbe refractometer. The density of the DNA after equilibrium had been reached was calculated either by the isoconcentration method (Vinograd, 1963) or by relating the density to that of a known market DNA. The values of fl were taken from Chervenka (1969). The strands of d(T-C-C),. d(G-G-A), were separated in a preparative alkaline CsCl density gradient; 6.9 OD280 units of d(T-C-C),.d(G-G-A),, 0.2 ml of 1 M NaOH, and 0.1 ml of 0.1 M EDTA were made up to 5.11 ml with water and 7.8 g of CsCl was added. The density was 1.805 g/ml. The solution was overlaid with Dow-Corning 556 fluid in polyallomer tubes, which were centrifuged at 35,000 rpm in the Ti 50 fixed angle rotor at 4" for 64 hr using the Beckman L2 65B ultracentrifuge. The gradient was pumped out into 16 fractions through a needle lowered into the tube, using a peristaltic pump to obtain a uniform flow rate. Fractions 1-8 contained d(G-G-A), and fractions 9-16 d(T-C-C),. The DNAs were dialyzed against 5 mM Tris (pH 8) and 0.05 mM EDTA. The ratio of the OD at 260-280 was 2.15 for d(G-G-A), and 1.28 for d(T-C-C),. The polymers were further characterized by RNA polymerase assays. Molecular Weights. Polymers were examined in the Spinco Model E ultracentrifuge, using the method and equations of Studier (1965). Nearest Neighbor Analyses of d(T-C-C),.d(G-G-A),. The standard polymerase assay conditions were used with one of the deoxyribonucleoside triphosphates labeled with 32Pin the a-phosphate. Reactions (50-111 scale) were terminated by adding a tenfold excess of EDTA over Mg2+ and sodium dodecyl sarcosinate to 0.5 %. Polymers were dialyzed free of triphosphates in a collodion bag (Sartorius) by repeatedly concentrating them with suction and adding water and un-
labeled dNTPs to chase out any remaining radioactivity not in DNA. Finally the DNA was taken up in 0.25 ml of 10 mM glycine (pH 10) and 2 mM CaC12, containing 0.2 pg/ml of micrococcal nuclease. After overnight digestion at 37 " most of the radioactivity was acid soluble. The solution was then made 100 mM in NH40Ac (pH 5.9) and spleen phosphodiesterase added to 50 pg/ml. After further incubation at 37' for 2 hr, about 0.5 ODzoounit of each of the four 3'-dNMPs were added to the solution. The solution was evaporated to dryness, taken up in the minimum volume of water (-50 pl), and electrophoresed on Whatman 3 mm paper at 30 V/cm for 3.5 hr in 0.1 M citrate (pH 3.5). The electropherograms were analyzed for radioactivity either by cutting them into 1-cm strips or else by running them through a radiochromatogram scanner. Nucleotide Analyses. The samples of DNA were degraded to the mononucleotide level with micrococcal nuclease and spleen phosphodiesterase as for the nearest neighbor analyses. When pancreatic nuclease and snake venom phosphodiesterase were used a contaminating enzymatic activity converted the nucleotides to other unidentified compounds. The mixture of nucleotides was separated and analyzed by high-pressure liquid chromatography using a Varian Aerograph LCS-1000 chromatograph equipped with a 3-m capillary column packed with Varian PA-38 anion exchange resin, essentially according to Brown (1970). The amounts of each nucleotide were calculated by comparing the peak areas, as determined by planimetry, with those of known amounts of authentic nucleotides. Approximately 1-2 nmol of total nucleotide was used per analysis. Results The Effect of the dNTP Concentration on DNA Synthesis. In order to obtain the maximum amount of DNA replication in a small volume it seemed that a high triphosphate level might be advantageous. Figure 1 shows the effect of varying the dNTP concentrations on the synthesis of d(T-G)n.d(C-A),. When each triphosphate is 0.2 mM, maximum synthesis occurs by about 90 min, and thereafter nuclease activity predominates. At a tenfold higher concentration, however, there is still active synthesis at 10 hr, and the degradative action appears to be inhibited. This inhibition may be due to the buildup of pyrophosphate concentration which would compete for OH- in the degradative reaction (Beyersmann and Schramm, 1968). At the higher triphosphate levels the level of Mg2+ must be increased. A good general rule is to use 5-7 mM excess Mg2+above the total triphosphate concentration. Thus at 2 mM in each dNTP (total 8 mM), a Mg2+concentration of 15 mM is used. If 5 mM Mg2+is used instead there is a complete lack of DNA synthesis, suggesting that triphosphates not bound to Mg2+are potent inhibitors of DNA polymerase. The net-fold synthesis of DNA can be as high as 100-fold in 5 hr, depending on the particular template and the initial DNA concentration. Correlation o j Ethidium Bromide Fluorescence and Acid Insolubility Assays for DNA. It has been shown that ethidium bromide fluorescence is specifically enhanced by duplex structures, and the sensitivity of the assay suggested it might be a suitable alternative to the acid insolubility or the hypochromicity assay. The latter assay is not feasible at the high triphosphate concentrations we use in any case. The disadvantage of the acid insolubility assay is the length of time taken from sampling to obtaining a value for the amount of DNA synthesized. The ethidium bromide fluorescence assay B I O C H E M I S T R Y , VOL.
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MINUTES 1 : The effect of dNTP concentration on d(T-G),.d(C-A), synthesis. The standard DNA polymerase conditions were used. Heated DIII (10 min, 97"), 25 pg/ml, 0.5 OD2soof unfractionated tRNA, 50 ng/ml of DNase I, and 0.206 ODzs0unit/ml of d(T-G),.d(C-A), were present. Each dNTP was present at the concentration indicated on the graph, with TTP 3H-labeled (2700 cpm/nmol) and dCTP I4C-labeled (2000 cpm/nmol); 10-pl samples were assayed by acid insolubility. FIGURE
provides an immediate value for the DNA synthesized and also gives further information on whether clc-DNA or d(A-T), .d(A-T), is being produced. In principle the ethidium bromide assay coupled with the acid insolubility assay can be used to determine if an appreciable amount of singlestranded DNA is present, since ethidium bromide measures only duplex structures, but acid insolubility measures total
DNA. As can be seen from Figure 2, the synthesis of d(T-G),. d(C-A), under several concentrations of triphosphates gave identical results when followed by acid insolubility or ethidium bromide fluorescence. This indicates that both strands are being synthesized in identical amounts, and this was confirmed by equal incorporation of 3H and 14C into the two strands. The input template is of course also measured in the ethidium bromide assay, but can be subtracted out. While radioactive incorporation can be made extremely sensitive with substrates of very high specific activity, in fact it is the size of the aliquots taken which will usually limit the repro-
28Xof col.voI.
dNTP's
FIGURE 2: Correlation of acid insolubility with the ethidium bromide fluorescence assay. The reaction mixtures and the samples were the same ones as used in Figure 1. The optical density of DNA synthesized was determined by the fluorescence of the test sample, compared to that of a known concentration of d(T-G),. d(C-A),.
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Agarose 5m gel filtration of d(T-G),.d(C-A), (see text).
DNA
SYNTHESIS
TABLE I :
Nearest Neighbor Analysis of d(T-C-C), . d(G-G-A),. Radioactivity in 3 '-Deoxymononucleotides
dTTP dATP dCTP dGTP
35,600 0 1,369 0
95.5 0 50 0
1,310 8 0 52,950
ducibility. Since in the later stages of synthesis 5/11 samples already contain far more DNA than is required for the ethidium bromide assay it is evident that little advantage is obtained from the potentally higher sensitivity of using radioactive substrates. Isolation of DNAs by Gel Filtration. Agarose gels provide a simple and quick procedure to isolate DNA free of protein and small molecules. Using a modification of an earlier report (Paetkau and Khorana, 1971), the reaction was stopped by the addition of excess EDTA (tenfold over MgZ+)and the solution made 0.05% in either sodium dodecyl sulfate or sodium dodecyl sarcosinate (Sarkosyl). Sarkosyl is often advantageous because potassium dodecyl sulfate is relatively insoluble. The solution was heated at 50" for a few minutes to ensure complete disruption of any DNA-enzyme complex. We routinely used Agarose 5m or 15m (50-100 mesh or finer), 0.8 cm2 x 31 cm, as shown in Figure 3 for the isolation of d(T-G),.d(C-A),. The eluting buffer in Figure 3 was 0.05 M triethylammonium bicarbonate (pH 8), which is readily removed by evaporation with ethanol. We routinely use 10 mM Tris-HC1 (pH 8)-0.1 mM EDTA, since the DNAs are stored in this buffer. Synthesis of d(T-C-C),. d(G-G-A),. After some preliminary variations of parameters the following procedure was adopted for the large scale synthesis of d(T-C-C),. d(G-G-A), from oligomers. Since polypurines are known to form secondary structures, the oligomers d(C-C-T)4 and d(G-G-A)3 (each 2.5 OD260units/ml) were heated to 100" for 2 min and cooled in ice in the absence of any added salts, before the rest of the reaction components were added. The fist 30 min of the reaction were at 22" (to prevent melting of the oligomers) and the temperature was then raised to 37" and fraction IV of the DNA polymerase preparation was added to a final concentration of 21 pg/ml. The reaction mixture (0.5 ml) contained 0.5 OD260unit of each oligomer. After 6 hr at 37" the reaction was terminated by addition of EDTA to 50 mM and sodium dodecyl sulfate to 25 mM. After heating at 50" for 5 min the DNA was isolated by Agarose 5m gel filtration as above, 5.5 ODgso units of d(T-C-C), d(G-G-A), appearing as a well-separated peak at the calculated void volume. This sample of DNA was then replicated on a small scale (50 pl) with [ITldCTP and r3H]TTP at the same time as d(TG),. d(CA), and d(TTC),.d(GAA),. The ratio of 1"C/3H incorporated into DNA was constant for each DNA during 9 hr of synthesis and the ratios were consistent with the base composition of the DNA. In Figure 4 the kinetics of synthesis for d(T-C-C),. d(G-G-A), are shown. The reaction was scaled up to 1 ml and after 9.5 hr at 37" the DNA was isolated as before; 0.5 OD260 unit of DNA was used as template with a final yield of 17.5 OD260units (35-fold synthesis). The DNA was checked for d(A-T), .d(A-T), contamination by transcription and contained about 0.75 % (see last section).
290 5,602 0 52,590
3.5 0 0 50
160 52 1372 0
0.8 99 0 50
0.4 0.9 50 0
Characterization of d(T-C-C),.d(G-G-A),. The most definitive criterion for the structure of repeating polymers is nearest neighbor analysis. This polymer was also characterized by transcriptional studies (Morgan, 1970), its buoyant density, the separation of the complementary strands in alkaline CsCl density gradients, the melting temperature, and nucleotide analyses. The nearest neighbor analysis of d(T-C-C), . d(G-G-A), is summarized in Table I. ~z-~~P-labeled TTP, dATP, dGTP, and dCTP were each used in turn to label the DNA. Occasionally the nearest neighbor analysis was not that expected theoretically for d(T-C-C),.d(G-G-A),, and this was due to a minor amount of contaminating d(A-T),. d(A-T), arising de novo in the reaction. A phenol extraction usually removed the d(A-T), d(A-T), (Laskowski, 1972) although it is irreproducible as a routine procedure in our hands. In Table I the radioactivity found in dAp and dTp after labeling with [ C X - ~ ~ P I ~ and T T P[ Q - ~ ~ P I ~ A T respectively, P, is almost certainly due to traces of d(A-T),.d(A-T),. In the [a-3zP]dGTP experiment about 0.5 of the radioactivity moved in the region of Tp on the electropherogram but did not coincide with it. It is most probably the nucleoside 3',5'-diphosphate from the 5'-end of the DNA which would not be further degraded by micrococcal nuclease and spleen phosphodiesterase.
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FIGURE 4: The kinetics of synthesis of d(T-C-C),.d(G-G-A),. The standard conditions for DNA polymerase were used with the following concentrations/ml: total dNTPs 8 m, with dCTP 14C labeled (5000 cpm/nmol) and ["H]dTTP (916 cpm/nmol), nucleotide on paper, d(T-C-C),. d(G-G-A), 0.55 ODzaounit, DNA polymerase fraction VI1 80 pg, fraction IV 96 pg and 15 mM MgClZ.Acidinsoluble [14C]dCTP(with restricted channels, no overlap of 3H) (X); ratio of 14C/3H counts (in restricted channels)( 0 ) .
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