Thermal renaturation of deoxyribonucleic acids

J. Mol. Biol. (1961) 3, 585-594

Thermal Renaturation of Deoxyribonucleic Acids t JULIUS MAR~IURt AND, PAUL DOTY

Department of Ohemistry, Harvard University, Oambridge, Massachusetts, U.S.A. (Received 29 March 1961) The conditions that produce the optimal re-formation of the native DNA conformation from denatured DNA have been examined. The restoration of the native conformation, called thermal renaturation, has been found to depend markedly on the source of the DNA; mammalian DNA coming from cells with very large DNA content renatures only slightly, bacterial DNA with greatly reduced DNA content per cell undergoes extensive renaturation, and the very smallest bacteria together with bacteriophage, having the lowest DNA contents, show nearly complete renaturation. With a given DNA, the optimal renaturation was found to occur at about 25° below the denaturation temperature, T m . The extent of renaturation was optimal above 0'4 M-Na+ and increased with molecular weight. The identity of the renatured DNA and the native material mID be shown in two ways: the similarity of the absorbance-temperature curves and the similarity of the rate of thermal inactivation of biological markers at temperatures somewhat above T m• This reproduceability of the helix -coil transition and the course of thermal inactivation demonstrates that the same secondary structure has re-formed and that non-specific hydrogen bonding is not involved.

1. Introduction

When DNA in aqueous solution is exposed to temperatures above the melting temperature, Pm (Marmur & Doty, 1959), the two strands in each molecule separate and assume randomly coiled configurations. Upon rapid cooling this denatured state is maintained, although considerable non-specific base pairing through hydrogen bonds occurs. However, if the thermally denatured DNA solution is cooled slowly, renaturation of the DNA takes place, resulting in a restoration of its transforming activity, physical, chemical and immunological properties (Marmur & Lane, 1960; Doty, Marmur, Eigner & Schildkraut, 1960; Levine, Murakami, Van Vunakis & Grossman, 1960).

The present communication is an attempt to describe the optimum conditions for the renaturation of DNA and to compare some properties of renatured and native DNA not previously described.

2. Materials and Methods Transformation techniques using Diplococcus pneumoniae as well as the method of obtaining the absorbance-temperature profiles have been previously described (Fox & Hotchkiss, 1957; Marmur & Lane, 1960; Doty et al., 1960; Doty, Boedeker, Fresco,

t This work was supported by a grant from the United States Public Health Service (C-2170).

t Present address: Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts, U .S.A. 585

586

J. MARMUR AND P. DOTY

Haselkorn & Litt, 1959). DNA was isolated from the following organisms according to the method of Marmur (1961): D. pneumoniae (R-36A); Bacillus subtilis (168); Escherichia coli (K-12 and B); Pseudomonas aeruginosa (NRRL B-23); the pleuropneumoniae-like organism, Mycoplasma gallisepticum (avian PPLO 5969); and from tobacco leaves. DNA samples isolated from salmon sperm and calf thymus were a gift from Dr. N. Simmons. DNA from the T-even E. coli bacteriophages was generously supplied by Dr. H. Van Vunakis. The DNA base compositions were obtained from published reports (Belozersky & Spirin, 1960; Chargaff, 1955) and confirmed by the T m of native samples (Marmur & Doty, 1959, 1961). The molecular weight of the native DNA samples used in these studies was approximately 10 ± 2 X 106 • Sonic degradation was carried out in the presence of the free-radical trapping agent AET (aminoethyl isothiuronium bromide hydrogen bromide) as described previously (Litt, Marmur, Ephrussi-Taylor & Doty, 1958).

3. Experimental (a) Effect of DNA source on renaturation

Although the T m for strand separation of DNAs having similar base compositions is independent of its source, the extent to which renaturation proceeds is greatly influenced by the type of DNA being studied. This becomes understandable when it is recognized that renaturation will depend on the concentration of specific complementary strands during the renaturation (Marmur & Lane, 1960). If there is no duplication of.sequences in the DNA molecules in a given cell and if the molecular weight of the isolated DNA is approximately the same, it is expected that the concentration of complementary pairs will fall off with increasing complexity of the species from which the DNA is isolated. Bacteria, which possess a relatively small number of nucleic acid molecules (Vendreley, 1958), contain a more homogeneous distribution of DNA molecules than that of DNA isolated from higher plants or animals but less homogeneous than the DNA isolated from bacteriophages (Crampton, Lipshitz & Chargaff, 1954; Brown & Brown, 1958). Thus, for a given concentration of DNA, renaturation would be expected to occur most readily with bacteriophage DNA and to be least complete with DNA from mammalian source such as calf thymus or salmon sperm. In order to explore this matter, renaturation was studied by following the absorbance at 260 mJ.L in the Beckman spectrophotometer. When DNA is denatured and rapidly cooled (quenched), approximately 60% of the bases are hydrogen bonded in a non-specific fashion, easily disrupted by exposure to temperature below the T m (Doty et al., 1959a). Upon raising the temperature to the region below the T m , annealing conditions are provided. These weak intramolecular hydrogen bonds are observed to rupture, by the increase in absorbance, and this as well as the elevated temperature allows the complementary strands the time and the opportunity to renature into double-stranded molecules which are hydrogen bonded and in register. Ifrenaturation occurs, the absorbance at 260 mJ.L will fall. Experiments of this type are shown in Fig. 1. It can be seen that after the melting of the weak, intramolecular hydrogen bonds of the denatured, fast-cooled DNA, renaturation of some of the DNA sample occurs. The rate and extent of renaturation of DNA is seen to be dependent upon its source. Calf thymus DNA (as well as DNA from salmon sperm and tobacco leaves) does not renature; D. pneumoniae DNA (as well as DNA from most bacterial sources) readily renatures, whereas the DNAs of T6r+ (as well as T2 and T4) and Mycoplasma gallisepticum (avian PPLO), which are more homogeneous (Hershey & Burgi, 1960; Guild, Morowitz & Castro, 1960; Guild, 1961), renature the most readily.

587

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(b) Optimum renaturation temperature: biological studies

Renaturation of DNA by slowly cooling a sample in a large water bath has led to somewhat variable results owing to the difficulty of cont rolling accura tely the rate of cooling. In order to find t he opt imum renaturati on t emperature of D. pneumoniae DNA, denatured samples were exposed at different constant temperatures for varying periods of tim e. The rate and extent of renaturation was followed eit her by the recovery of t ransforming activity or by the decrease in absorbance. RENATURATION

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FIG. 1. Thermal trans it ions of denatured D NA sa mples . DNA at 20 pg/mI. was hea ted a t 100 °0 for 10 m in in 0·3 ~1-NaCI plus 0·03 M-Na citrat-e and q uickl y cooled. The sa m ples of D NA (in ground glass stop pere d cuvettes ) at the same ca n . cent ra t ion and in the sa me solve nt, wer e then pl ac ed in t he Beckman spec t ro photometer cha m be r , prewarmed to 67 °C. The initial part of t he gr aph represen t s t he increase in absorban ce at 260 ml-' as t he dena tured D NA samples reach te m pe rature equilibri um. The curves to t he right of t he vertical lin e rep resent t he decreas e in a bso rbance of the te mpe rature .equ ilibrated, renaturing samples as a function of time of exposure a t 67°0. DNA was isolated fr om t he organism s listed in the Figure.

When the course of renaturation of D. pneumoniae DNA is followed by the restoration of the ability to transform with respect to the streptomycin resistance marker, the results are found to depend very much on the temperature. As shown in Fig. 2, the recovery of biological activity displays an initial rapid rate, followed by a slower one. At the optimum temperature, the slow increase continues for at least 5 hr. When a plat eau is reached in the restoration of the biological activity, subsequent cooling to room temperature result s in an addit ional increment in transforming activity (10 to 50%) depending on t he previous temperature. By exposing denatured DNA to the opti mum renaturati on t emperature for 2 to 3 hr, as well as a subsequent slow cooling to room temperature, restorati on of 40 to 50% of the original biological activity is generally obtained in the case of D. p neumoniae DNA. These conditions may readily be used for t he renaturati on of DNA which has suffered denaturati on by means other than t hermal exposure, such as formamide (Marmur & Ts'o, 1961); t hus a mild meth od for renaturation, which avoids exposure to relatively high te mperat ure, is available.

J. MA R:\lUR AND P . DOTY

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In order to find the optimum te mperat ure for t he restoration of the transforming acti vity of'D. p neumoniae DNA at the ionic st rengt h employed, the initial rates of ConcQ DNA:10fLg/mJ.

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F IG. 2. Effect of temperature on t h e recovery of streptomycin-transfo rm ing activity of therma.lly denatured D . pneumonia e DNA. DNA from streptomycin resist ant cells of D. pneumoniae was denatured a.t 10 p.g /ml. by heating for 10 min at 100 0 e in 0·3 M·NaCI plus 0·03 M·Na cit rate. Portions, at the same DNA and sol. vent concent rat ion, wer e then dist ribu ted and exposed to various tempera t u res . Samples were removed and quickl y cooled at differen t ti mes an d as sayed for st reptomycin .t ransforming act ivit y .

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589

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recovery of the biological activity were plotted (Fig. 3) against the renaturation ternperature. The optimum temperature lies within the range 65 to 70°0. The slightly higher value obtained by this method, compared with that obtained by the absorbance method (see following section), may be a reflection of the observation that the streptomycin resistance marker (used in the biological assay for the renaturation) is probably associated with a DNA molecule which has a higher guanine plus cytosine content than the average composition of the total DNA population (Marmur & Lane, 1960). (c) Optimum renaturation studies: absorbancy studies

It has been shown (Fig. I) that the renaturation of DNA can be followed by the decrease in absorbance after the intramolecular hydrogen bonds of denatured, fastcooled DNA are melted out. When the initial rate of decrease in the absorbance is plotted against the renaturation temperature, the curve shown in Fig. 4 is obtained. The optimum renaturation temperature for D. pneumoniae DNA is 65°0.

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(d) Effect of base composition on the optimum renaturation temperature

Using the absorbance method, the optimum renaturation temperature for DNA from various bacterial sources was determined. When the optimum temperature was plotted against the guanine plus cytosine content of the DNA, the results shown in Fig. 5 were obtained. As the guanine plus cytosine content increases, there is a gradual increase in the optimum renaturation temperature. (e) Effect of molecular weight

In order to compare the rate and extent of the renaturation of denatured DNA as a function of the molecular weight, sonically degraded D. pneumoniae DNA of molecular

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weight was compared with DNA of molecular weight 10 X 106 • The degraded material retained 0·24% of it stransforming activity with respect to t he streptomycin resistance marker. The renaturation of the rmally denatured samples of both the

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undegraded and sonically treated DNA samples was studied by the restoration of tho biological activity under optim um conditions of temperature and ionic strengt h. In Fig. 6 are shown t he differences in t he rate and exten t of renaturat ion of the two samples. The transforming DNA was first denat ured by heati ng t he samples in

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0·3 III-NaCI plus 0·03 M-Na citrate at lOO°C for 10 min and then incubated at 65°C at 15fig perml. in the same solvent. Samples,removed at various times and quickly cooled, were assayed for t he ability to transform sensitive cells of D . pneumoniae to streptomycin resistance. It can readily be seen that both the rate and extent of the recovery of biological activity are dependent on the molecular weight. Similar results are obtained when renaturation is followed by the absorbance-temperature method. Experiments summarized in Fig. 7 again clearly show that th e re-formation is more rapid and extensive the higher the molecular weight. In t his experiment, heat denatured, fast -cooled sampl es in ground glass st oppered cuvettes were placed in t he 36 32

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Beckman spectrophot ometer chamb er prewarmed to 66°C. When the intramolecular hydr ogen bonds had melted, as measured by t he increase in absorbance, the renaturat ion, indicated by t he subsequ ent decrease in absorbance, was followed for both samples. After maximum re-formation had occurred, the percentage re-formation was plotted as a function of time of exposure to 66°C. (f) T hermal stability of renatured DNA

If renat uration is indeed the restoration of hydro gen bonding between complement ary st rands whose bases are in register , then the t herma l stability of renat ured DNA should mimic t hat of native DNA. This was tested by exposing both renatured and native DNA to a temperature several degrees above the T m and following their transforming activity as a function oftiroe. This has been shown previously (Doty , Marmur & Sueoka, 1959)to result in an easily measurable, time-d ependent loss of transforming activity due to denaturation of the molecules carrying the genetic marker. Renatured DNA was prepared as follows: D. pne umoniae was heated at 100°C for 10 min in 0·3 M-NaCl plus 0·03 ~I-Na cit rate and then exposed to 65°C at 20 fig/ml. in the same solvent for varying periods of time. Samples were removed at several times, quickly cooled, and dialy sed against 0·15 III-NaCI plus 0·015 III-Na cit rat e.

592

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The thermal inactivation curves at 89·2°C in 0·15 M-NaCI plus 0·015 M-Nacitrate of the native and renatured dialysed samples are shown in Fig. 8. DNA renatured for 3hr (41% restoration of biologicalactivity) as well as samplesrenatured for 25min (33% restoration) and 60 min (36% restoration) showed the same thermal stability as did native DNA. Data for the latter two renatured samples are not shown in the Figure. DNA renatured for 4 min (7% restoration of biological activity) shows an abnormally higher thermal resistance which may be a reflection of the presence of a resistant component selected during the initial (100°0) thermal exposure. 100.---------------,

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It is suggested that this (or the absorbance-temperature profile) criterion of the thermal stability of the material should be used as a test of its native-like structure to rule out the possibility of non-specific aggregation. The absorbance-temperature profile can also be employed as a criterion to check the structure of renatured DNA. The profiles of native and denatured DNA differ significantly (Doty et al., 1959a). Whereas native DNA melts very sharply, resulting in a steep rise in optical density in the T m region, denatured DNA gives rise to a broad profile. Because of the high degree of homogeneity of the DNA from phage and PPLO, an attempt was made to see to what extent DNAs from these sources could be renatured and to examine their thermal stability. T6r+ DNA and avian PPLO DNA were denatured and then renatured under optimum conditions in 0·3 M-NaCI plus 0·03 M-Na citrate. Each was then compared to its corresponding native preparations for its thermal stability in the same solvent. From Fig. 9(a) and (b) it is clearly seen that a large portion of each pair of curves can be superimposed in the T m region and can be readily accounted for, if one assumes that 70 to 80% of the denatured molecules have renatured when exposed to the optimum renaturation conditions. Moreover, we have recently found (Cordes & Marmur, unpublished observations) that denatured DNA

RENATURATION OF DNA

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4. Discussion By studying the renaturation of various samples of DNA we have been able to confirm previous notions about the properties of DNA and to advance our understanding of its structure. Thus, the inability to renature DNA from higher plants and animals is predictable because of their much greater heterogeneity and the dependence of renaturation on the concentration of specific strands. DNA from relatively homogeneous sources (e.g. bacteriophage) are expected to and indeed do renature at the greater rate and to a higher extent. It remains to be shown whether or not, by fractionating DNA from heterogeneous sources, renaturation of selected, more homogeneous fractions can be attained. The optimum renaturation temperature is approximately 25°C below T m- The reason for this may be the result of several factors . An elevated temperature is required for the melting of the weak, intramolecular hydrogen bonds of the fastcooled, denatured DNA strands as well as to disrupt any mismatched, non-complementary strands. It would be expected that the recognition of complementary strands requires a certain minimum number of base pairs which are large enough in number to resist being melted out at the elevated, optimum temperature. Once the minimum nucleation region has been achieved, the remaining bases of the complementary strands, which are in register, can be imagined to "zip" up to complete the renaturation of the molecule. DNA with a higher guanine plus cyt osine content would be expected to have a higher optimum renaturation temperature. Employing optimum conditions, denatured fast -cooled D . p neumoniae DNA (with approximately 0·5% residual biological activity) can be renatured to 50 and sometimes 60% of the original transforming activity. The inability to achieve complete renaturation is due to some phosphate-ester backbone breakage of the DNA strands during the denaturation and renaturation (Doty et al., 1960) and perhaps also to depurination (Greer & Zamenh of, 1959) as well as to the presence of some residual

594

J. MARMUR AND P. DOTY

unrenatured molecules. Employing milder denaturation conditions (e.g. formamide) and a more homogeneous DNA source, it would be expected that, under optimum renaturation conditions of DNA concentration, ionic strength and temperature, higher degrees of renaturation will be attained. The authors wish to thank Mr. C. Schildkraut for his advice and suggestions and Miss D. Lane for participating in the biological experiments.

REFERENCES Belozersky, A. N. & Spirin, A. S. (1960). In The Nucleic Acids, ed, by E. Chargaff & J. N. Davidson, Vol. 3, p. 147. New York: Academic Press. Brown, G. L. & Brown, A. V. (1958). Symp. Soc. Exp. Biol. 12, 6. Chargaff, E. (1955). In The Nucleic Acids, ed. by E. Chargaff & J. N. Davidson, Vol. 1, p. 521. New York: Academic Press. Crampton, C. F., Lipshitz, R & Chargaff, E. (1954). J. Biol. Ohem, 211, 125. Doty, P., Boedtker, H., Fresco, J. R., Hasolkorn, R. & Litt, M. (1959a). Proc, Nat. Acad. Sci., Wash. 45, 482. Doty, P., Marmur, J., Eigner, J. & Schildkraut, C. (1960). Proc. Nat. Acad. Sci., Wash. 46,461. Doty, P., Marmur, J. & Sueoka, N. (1959b). In Structure and Function of Genetic Elements, p. 1. Brookhaven Symposia in Biology. Fox, M. S. & Hotchkiss, R D. (1957). Nature, 177, 1322. Greer, S. & Zamenhof, S. (1959). Fed. Proc. 18, 939. Guild, W. R (1961). Fifth Annual Meeting of the Biophysical Society, FB 9. Guild, W. R, Morowitz, H. J. & Castro, E. (1960). Fourth Annual Meeting of the Biophysical Society, p. 19. Hershey, A. D. & Burgi, E. (1960). J. Mol. Biol. 2, 143. Levine, L., Murakami, W. T., Van Vunakis, H. & Grossman, L. (1960). Proc. Nat. Acad. Sci., Wash. 46, 1038. Litt, M., Marmur, J., Ephrussi-Taylor, H. & Doty, P. (1958). Proc, Nat. Acad. Sci., Wash. 44, 144. Marmur, J. (1961). J. Mol. Biol. 3, 208. Marmur, J. & Doty, P. (1959). Nature, 183, 1427. Marmur, J. & Doty, P. (1961). In preparation. Marmur, J. & Lane, D. (1960). Proc. Nat. Acad. Sci., Wash. 46, 453. Marmur, J. & Ts'o, P. O. P. (1961). Biochim. biophys. Acta, in the press. Vendreley, R (1958). Ann. Inst. Pasteur, 94, 142.