Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part II, pp. 3834-3838, December 1973

Reverse Transcription by DNA I (hybrid templates/poly(A) * poly(U)/poly(C) * poly(I)/"fl RNA") JOHN D. KARKAS Cell Chernistry Laboratory, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032 Communicated by -Erwin Chargaff, August 13, 1973

ABSTRACT E. coli DNA polymerase I (EC 2.7.7.7) can MATERIALS AND METHODS engage in either DNA- or RNA-directed DNA synthesis with hybrid templates. The choice of the strand to be . DNA polymerase I (EC 2.7.7.7) was prepared transcribed depends primarily on the relative lengths of from E. coli (6, 7). The preparation had a specific activity the two strands of the hybrid, the longer strand serving as (defined and determined as in ref. 7) of 15,600 units/mg of the template and the shorter as the primer. If a poly- is reduced in size by exposure to an endo- protein. RNA polymerase (EC 2.7.7.6) was isolated from E. nuclease before being hybridized to the complementary coli by a modification (J. G. Stavrianopoulos, unpublished) strand, the template efficiency of the latter increases of a recent procedure (8). Terminal deoxynucleotidyl trans- several-fold. Under properly selected conditions, highly ferase was prepared from calf thymus as described (9, 10). efficient reverse transcription of the all-ribonucleotide template-primers poly(A) .oligo(U), poly(C) .oligo(I), and Sheep-kidney nuclease, an endonuclease producing oligonu- poly(I) *oligo(C) can be achieved. "fl RNA," the RNA cleotides with 5'-PO4 and free 3'-OH ends, was isolated as strand of an fl DNA RNA hybrid, can also serve as tem- described (11), with minor modifications. Highly purified plate for reverse transcription either after "nicking" of calf-thymus RNase H (12) was a gift of Dr. J. G. Stavriano- the hybrid with DNase, or after separation from the DNA poulos. Pancreatic DNase (EC 3.1.4.5), electrophoretically strand and priming by DNase-treated fl DNA. purified, was purchased from Worthington Biochemicals. RNA-directed DNA synthesis or "reverse transcription" was originally believed to be a unique property of the Polynucleotides. Poly(dA) and poly(dT) were prepared of certain RNA viruses. It was subsequently demonstrated in with terminal deoxynucleotidyl , as described several laboratories, including our own (1-5), that various (13), using as initiators the corresponding tetranucleotides, "normal" DNA polymerases are capable, at least under purchased from Collaborative Research, Oligo(dT)12_18 was a specific conditions in vitro, of transcribing the RNA strand of product of P-L Biochemicals. Poly(A), poly(C), poly(U), and suitable template-primer combinations; the template-primers poly(I) were purchased from Miles Laboratories. Polynu- usually used were hybrids of synthetic homopolymers and cleotides treated with nuclease are referred to as oligonucleo- were most often poly(A) * poly(dT) or poly(A) - oligo(dT). tides in this communication. We recently reported the use of hybrid template-primers fi DNA was isolated from bacteriophage fi by phenol ex- by a-DNA polymerase isolated from chicken embryo and traction and purified by gel filtration through Agarose (Bio- purified to homogeneity (1, 3, 5) and by highly purified DNA Gel A-1.5m); only the DNA eluted within the exclusion polymerase I of Escherichia coli (2, 4). The synthetic homo- volume was collected. The fi DNA. [14C]RNA hybrid (1:1) polymer poly(A) -poly(dT) was the most efficient of all tem- was prepared and purified as described (5). The "fl RNA" plate-primers tested for both of these enzymes. With this was isolated from this DNA * RNA hybrid by one of the follow- template-primer, poly(dT) was the product; dATP incorpora- ing two methods: (a) The hybrid, dialyzed against 1 mNI tion was minimal. This result would seem to indicate that NaCl, was melted by heating at 100° for 10 min, quickly RNA-directed DNA synthesis was the preferred mode of cooled, and immediately applied to an Agarose column (Bio- action even for E. coli DNA polymerase I. However, when Gel A-1.5m) at 4°. Upon elution with 1 mM NaCl, the DNA, "natural" hybrids were used as templates, such as the one still bound to 20% of the radioactive RNA, was eluted first produced by transcription of phage fi DNA with RNA poly- as a sharp peak within the void volume; it was followed by a merase, the synthesis of DNA was DNA-directed: the com- shallow peak of RNA, identifiable by its ratio of UV absorb- position of the DNA product was complementary not to that ance to radioactivity; 10% of this radioactivity became of the RNA strand of the hybrid but to that of the fi DNA soluble in trichloroacetic acid upon treatment with RNase (4, 5). It was, therefore, evident that DNA polymerase I, H. (b) The hybrid (250 'g) was adsorbed on a small (400 given a hybrid template, could engage in either DNA- or ,ul of bed volume) column of hydroxylapatite; a 2-ml solution RNA-directed synthesis. In a recent communication (5) we of 1 mg of DNase in 0.05 M K2HPO4-10 mM MgCl2 was proposed that the choice of the strand to be copied is deter- percolated slowly through the column. After washing with mined primarily by the relative lengths of the two strands of 0.05 M K2HPO4, the radioactivity was eluted with 0.5 ml of the potential template-primer, the longer becoming the tem- 0.5 M K2HPO4. (I thank Dr. J. G. Stavrianopoulos for sug- plate and the shorter serving as the primer. gesting this procedure.) The material was then applied to a The experiments presented here offer direct experimental Sephadex G-100 column (2.5 X 50 cm) kept at 750 and eluted, support for this proposal, and continue the exploration of the at this temperature, with 1 mM NaCl. The RNA appeared conditions under which DNA polymerase I of E. coli can en- as a sharp peak within the void volume, and had the expected gage in RNA-directed DNA synthesis. ratio of UV absorbance to radioactivity. 3834 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) Reverse Transcription by DNA Polymerase 3835

Precursors. Ribonucleoside triphosphates were from Pharma Waldheim (West Germany); deoxyribonucleoside triphos- phates from Sigma; 14C-labeled ribonucleoside triphosphates and 3H-labeled deoxyribonucleoside triphosphates from Schwarz/Mann and New England Nuclear Corp. RESULTS AND DISCUSSION Relative Size of the Strands. When commercial preparations of poly(A) poly(dT) are used as template-primers for E. coli DNA polymerase I (1, 2), high levels of incorporation are recorded with dTTP as the precursor, but very low or no in- corporation when dATP is the precursor. This apparent pref- erence for the ribonucleotide strand as the template could be explained on the basis of the different lengths of the two strands in such preparations: poly(A), synthesized by poly- nucleotide , has an s20 value of 7-8, while poly (dT) is usually prepared with terminal deoxynucleotidyl transferase and has an s20 value of 2.5-3. One can, therefore, assume that in the hybrid several pieces of poly(dT) are aligned on each molecule of poly(A), the gaps between them becoming primer sites for the polymerase. The opposite pref- erence would be observed should the poly(dT) molecules be longer that those of poly(A). This hypothesis is confirmed by the experiment of Fig. 1A, in which the poly(A) was first subjected to treatment with sheep-kidney nuclease for various lengths of time before being added to the poly(dT) and in- cubated with DNA polymerase. The of FIG. 1. Effect of nuclease treatment of one strand on the incorporation dTMP, template-primer properties of polynucleotide pairs. The poly- very high with untreated poly(A), drops quickly as the poly nucleotide (3.6 mg) was incubated with 40 units of sheep-kidney (A) is reduced in size by the action of the nuclease. On the nuclease in 4 ml of buffer solution (50 mM Tris.HCl, pH 7.7- contrary, dAMP incorporation, insignificant in the beginning, 1.5 mM MgCl2-5 mM 2-mercaptoethanol). At the times indicated, increases to appreciable levels with increasing time of incuba- 0.5-ml aliquots were removed and heated at 700 for 10 min to tion of the poly(A) with nuclease; after 120 min of incubation, inactivate the nuclease. Ten microliters (9 Mg) of each sample of the picture is almost entirely reversed, poly(dA) being now nuclease-treated polymer was mixed at room temperature with synthesized at a rate several times higher than that of poly- 10 Mul of a solution (1 mg/ml) of untreated complementary poly- (dT) synthesis. The level of dAMP incorporation never nucleotide. After 5-15 min, the template-primers were incubated reaches that of dTMP incorporation obtained with untreated with DNA polymerase I. The incubation mixtures contained, in a poly(A), most probably because the initial of the final volume of 0.13 ml: 50 mM Tris .HCl, pH 8.3, 0.5 mM MnCl2, length 0.1 M KCl, 0.15 mM of either [3H]dTTP or [3H]dATP (20 poly(A) is much higher than that of the poly(dT)*. cpm/pmol), and 125 ng of the polymerase. After 30 min of This shift of the polymerase in terms of its preferred tem- incubation at 280, the reaction was terminated with 1.5 ml of plate from one strand to the other can be observed not only cold 10% trichloroacetic acid; the precipitates, collected and with hybrid template-primers but also when both strands are washed with 5% trichloroacetic acid on membrane filters deoxyribo- or both ribopolynucleotides. Thus, with the pair (Schleicher & Schuell, Type B-6), were counted in dioxane- poly(dT) poly(dA), dTMP incorporation drops and dAMP naphthalene scintillation mixture, in a Beckman LS-250 counter. incorporation increases as the size of the poly(dA) decreases 0, dAMP incorporation; 0, dTMP incorporation. (A) poly(dT) by treatment with nuclease (Fig. 1B). Qualitatively similar and nuclease-treated poly(A); (B) poly(dT) and nuclease- results are obtained with the ribonucleotide-ribonucleotide treated poly(dA); (C) poly(U) and nuclease-treated poly(A). pair poly(U) -poly(A) as the poly(A) is reduced in size (Fig. 1C), although incorporation remains at a lower level. The importance of the size of the primer for the efficiency of poly(A) or poly(dA) and the primers poly(U) or poly(dT), incorporation directed by the complementary template exposed for various lengths of time to the action of sheep- strand is further demonstrated by the experiments summa- kidney nucleaset. Only the incorporation of dTMP is illus- rized in Fig. 2. The template strands in this series were either trated in Fig. 2, since that of dAMP was insignificant. With untreated poly(U) as the primer, dTMP incorporation is

* very low with both poly(A) and poly(dA) templates; as the What appeared as a discrepancy between our findings with nuclease DNA polymerase I and poly(A)-poly(dT) and those of Cham- treatment of the poly(U) proceeds, dTMP incorpora- berlin with the same system (15) can now be explained in terms of tion increases, eventually reaching a level 50 times higher the relative lengths of the two strands in the templates used. In our poly(A) -poly(dT) the s20 values of poly(A) and poly(dT) t Sheep-kidney nuclease was used in all of the experiments re- were 7.8 and 2.75, respectively, while Chamberlin used a poly- ported here for uniformity, since this nuclease attacks both ribo- (dT) of higher sedimentation (s20 13.9) than that of his poly(A) and deoxyribopolynucleotides. When the polynucleotide to be (820 8.7). Thus, in our template it was the ribonucleotide strand used as primer is of the deoxyribo type, very similar results are that was the longer one, and hence more suitable for transcrip- obtained with pancreatic DNase replacing the sheep-kidney tion, whereas the opposite was true for Chamberlin's template. . Downloaded by guest on September 26, 2021 3836 Biochemistry: Karkas Proc. Nat. Acad. Sci. USA 70 (1973)

E O.EU £2.0 z E 1.5 0 0.4 / | | 9 < /I% , l.0 5~ ~ %o 0 o\T z a. 0.5 W 0.2 ° 0 -~~~~~~~~~-4--. U 0 z

3U 60 9O 120 15O KO --- c. 6 A ------MINUTES OF INCUBATION WITH NUCLEASE -4 " 0 FIG. 3. Changes in the template efficiency of poly(C) and poly(l) upon nuclease treatment of the complementary primer 27 strand. Experimental conditions for the nuclease treatment and the polymerase assay as in Fig. 1, with the exception of the KCl

30 60 90 20 10 BO concentration, which was reduced to 0.05 M. The hybridization MINUTES OF INCUBATION WITH NUCLEASE step was performed at 700, as described in Table 1. 4.6 ug of FIG. 2. Changes in the template efficiency of poly(A) and template-primer (2.5 gg of polynucleotide and 2.1 jug of oligo- poly(dA) upon nuclease treatment of the complementary primer nucleotide) were used per assay. 0, dGMP incorporation with strand. (A) Nuclease-treated poly(U) primer; (B) nuclease- poly(C) and nuclease-treated poly(l); 0, dCMP incorporation treated poly(dT) primer. Experimental conditions as in Fig. 1, with poly(I) and nuclease-treated poly(C). except that in the poly(A)-poly(dT) pair, the amounts of tem- plate and primer were reduced: 1.7 ,/g of poly(A) and 1.6 jg of poly(dT) were used per assay. Solid line, poly(dA) template; purified DNA polymerase and poly(A) - poly(U) as the template. broken line, polytA) template (14). It was suggested, however, that this reaction proceeds by a semiconservative mechanism involving the than that obtained with untreated poly(U) (Fig. 2A). The initial formation of hybrids, the DNA strands of which serve level of incorporation seems to depend on the size of the as the real templates thereafter (15). Such mechanism cannot oligo(U) and not on the nature of the template strand, since be invoked, of course, when dTTP is the only precursor (see the same level is obtained with poly(A) and poly(dA) as Fig. 2) with poly(A) templates. In this case, poly(dT) has to templates. With nuclease-fragmented poly(dT) as primer, be made by reverse transcription of the poly(A) strand. This one can again observe an increase in dTMP incorporation seems to occur with high efficiency whether the primer is upon nuclease treatment of the primer (Fig. 2B). This time, ribo- or deoxyribonucleotide (Fig. 2A and B, respectively), however, the efficiencyof the two templates is not the same: in- provided that it is of the same length. The poly(U) strand, corporation reaches the same level in Fig. 2B, but (see legend) however, does not seem to be an efficient template for reverse the amounts of template and primer used for poly(A) were transcription with either deoxyribo- or ribonucleotide primers. much lower than in that of the poly(dA) t. When the same This is seen by the low incorporation of dAMP obtained in the amounts of template-primer are used and the primers are of experiment of Fig. 1C and in a similar experiment where oligo- the same size, poly(A) is a much more efficient template than (dA) replaced oligo(A) as primer with equally poor results. poly(dA). One possible reason for this difference is the differ- The fact that reverse transcription by E. coli DNA poly- ent length of the two polymers used here: poly(A) was longer merase I is not a peculiarity encountered only for poly(A)- than our poly(dA). There may, of course, be other reasons re- directed poly(dT) synthesis is demonstrated with the next garding the relative strengths of interaction among template, few experiments involving the use of poly(C) and poly(I) as primer, and product. templates and the complementary polynucleotides, frag- An attempt was made to evaluate the average chain length mented with sheep-kidney nuclease, as primers. It is possible, of the nuclease fragments of poly(dT) by comparing the in- with these systems, to achieve relatively high levels of reverse corporation obtained with poly(A) as template and oligo- transcription, but (Tables 1 and 2 and Fig. 3) the require- (dT)12_18 as primer to that recorded with the same template ments for these reactions are more stringent as regards the and nuclease-treated poly(dT) samples as primers, in an ex- size of the oligonucleotide primer, the conditions of hybridiza- periment similar to that of Fig. 2B. Incorporation with the tion, and the concentration in the incubation mixtures of both oligo(dT)12..l8 primer was higher than that obtained with the salt and metal ions. One can see, for instance, with the poly- nuclease-fragmented poly(dT) even after 4 hr of treatment of (C) oligo(I) pair (Table 1), that dGMP incorporation drops the poly(dT) with the nuclease. Fragmentation of the poly- sharply if the molarity of Mn2+ increases slightly or if Mg2+ (dT) does not seem to be extensive under these conditions. replaces Mn2+. This is not the case with poly(A) * poly(dT): Reverse Transcription of Ribohomopolymers. One could with this template about equal levels of dTMP incorporation are mM Mn2+ or 3 mM at and consider as the first example of reverse transcription on recorded with 1 Mg2+ pH 8.3, are between and 2 record the synthesis of poly(dT) -poly(dA) with a partially very minor differences observed 0.5, 1, mM Mn2+ (Fig. 1 of ref. 2). Table 1 also shows (last line) that if the hybridization is not performed at 70° with slow cooling The use of a low template concentration becomes necessary with $ but at room poly(A) .poly(dT), in order to demonstrate the increase in dTMP by simple mixing temperature, incorporation incorporation as the size of the poly(dT) is reduced. With higher drops by about 70%. levels of template-primer, incorporation is already very high with Table 2 demonstrates the importance not only of the diva- untreated poly(dT) and a plateau is reached within the first few lent metal but also of the molarity and nature of the salt minutes of nuclease treatment. present during incubation. A molarity of 0.05 seems to be Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 70 (1978) Reverse Transcription by DNA Polymerase 3837

TABLE 1. Effect of metal ion concentration and hybridization TABLE 2. Effect of metal ion and salt concentration on temperature on the template efficiency of poly(C) .oligo(I) the template efficiency of poly(I) -oligo(C) Temperature dGMP dCMP Concentration of incorporation incorporation Metal (mM) hybridization (pmol) Metal Salt (pmol) Mg 6 700 18 Mg (4 mM) NaCl (0.02 M) 58 Mg 3 700 28 Mn (1 mM) NaCl (0.02 M) 149 Mn 2 700 24 Mn (0.5 mM) NaCl (0.02 M) 278 Mn 1 700 261 Mn (0.5 mM) NaCl (0.05 M) 679 Mn 0.5 700 794 Mn (0.5 mM) NaCl (0.10 M) 545 Mn 0.5 250 269 Mn (0.5 mM) KCl (0.05 M) 684 Mn (0.5 mM) KC1 (0.10 M) 246 Poly(I) was incubated for 1 hr with sheep-kidney nuclease Mn (0.5 mM) KC1 (0.20 M) 14 under the conditions described in Fig. 1. Hybridization with poly(C) was performed as described (16); aliquots of the nuclease- Poly(C) was incubated for 20 min with sheep-kidney nuclease treated poly(I) (0.85 mg/ml in 0.01 M KPO4, pH 7.0-0.15 M under the conditions described in Fig. 1 and the resulting oligo(C) NaCl) and of untreated poly(C) (1 mg/ml) were heated separately was hybridized with untreated poly(I) at 700, as described in at 700 for 10 min, mixed at 700, and kept at this temperature for Table 1. For each polymerase assay, 4.6 Ag of the hybrid [2.5 another 10 min. The heating element of the water bath was then sg of poly(I)-2.1 ,g of oligo(C)] was used; conditions as in Fig. turned off, whereupon the solutions returned to room temperature 1, except for the metal and salt concentrations, which were as very slowly (about 15 hr). For the experiment of the last line, indicated. the solutions were simply mixed at room temperature. Five microliters of the solution of the hybrid [2.5 Asg of poly(C)- the product, was used here to demonstrate reverse transcrip- 2.1 ug of oligo(I)] was used for each polymerase assay performed tion with "f1 RNA" as the template (Table 3). In one ex- as in Fig. 1, except for the metal ion concentration, which was as indicated. TABLE 3. Base composition offi DNA, "fi RNA", and of the products of DNA- and RNA-directed synthesis by optimal for dCMP incorporation; moreover, at 0.1 M a differ- E. coli DNA polymerase I ence between NaCl and KCI can be observed (lines 5 and 7). Base composition (mol/100 mol) Fig. 3 illustrates the results of an experiment similar to those of Figs. 1 and 2. As for A, T, and U polymers, nuclease DNAor RNA A G C T or U Ref. treatment of the primer results in an increase in incorpora- fl DNA 24.5 21.1 21.3 33.1 (4) tion; However, as the nuclease exposure proceeds, the initial fl DNA 24.7 20.3 20.8 34.2 (17) increase is followed by a decline, which is particularly sharp "fi RNA" 33.6 22.0 19.6 24.8 (5) for the poly(I) oligo(C) pair. There is, in this case, a rather DNA product narrow range of sizes of oligonucleotides that can serve as (Exp. 1) 33.8 20.8 20.0 25.5 This work primers for DNA polymerase. This, most probably, reflects (Exp. 2) 26.2 21.6 18.3 33.9 This work the inability of very small oligonucleotides to form stable (Exp. 3) 26.3 19.8 19.9 34.0 This work complexes with the complementary polynucleotide, at least (Exp. 4) 26.8 22.0 17.4 33.8 This work at the ionic strength and temperature of incubation used. The experiments described, however, leave no doubt that For each determination of the base composition of the DNA under carefully chosen conditions, E. coli DNA polymerase synthesized by DNA polymerase, four identical polymerase I can synthesize poly(dC) and poly(dG) by reverse transcrip- assays were performed with all four deoxyribonucleoside tri- tion of all-ribonucleotide template-primers with phosphates present but a different one labeled in each case. relatively Both labeled and unlabeled precursors were purified by chro- high efficiency. matography on Dowex-1. The specific activity of the labeled Reverse Transcription of Heteropolymeric RNA. In the precursors was estimated by triplicate determinations of their reverse transcription experiments described so far, both the UV absorbance and radioactivity. From the incorporations of the individual bases, the of DNA was polynucleotide template strand and the ribo- or deoxyribo- composition the product computed. Each analysis was performed in triplicate. The com- were nucleotide primers homopolymers. It would be desirable, position of the polymerase assay mixtures and other conditions however, to demonstrate reverse transcription also with were as described in Fig. 1, except for the presence of all four heteropolymeric RNA templates, since with such templates precursors and the lower salt molarity (0.05 M KCl). The follow- the possibility of a "slippage" mechanism or other similar ing template-primers were used: Exp. 1: (DNA-directed syn- artifacts is very much reduced. The difficulty encountered thesis) 2 ug of intact fi DNA-RNA hybrid per assay. Exp. 2: with heteropolymeric templates, in contrast to the homo- 32 ug of f I DNA * RNA hybrid was incubated with 2 ug of DNase polymers, is to prove that the product is complementary to in 0.6 ml of 0.05 M Tris HCl, pH 7-4 mM MgCl2 for 15 min at the template strand and not to the primer, if the latter is 370 and then heated at 800 for 10 min to inactivate the nuclease; also heteropolymeric. With the fl DNA-RNA hybrid we 2 ug of this "nicked" hybrid was used in each polymerase assay. have shown by "transcription analysis" (4, 5) that the base Exp. 3: 100 ug of fi DNA was incubated with 40 yg of DNase in 0.5 ml of 0.05 M Tris*HCl, pH 7-4 mM at composition of the product was MgCl2 for 15 min complementary to that of the 370; the nuclease was then inactivated by heating at 1000 for DNA strand of the hybrid, taking advantage of the fact that 15 min; 0.3 ug of this "primer" DNA and 0.7 jg of "fi RNA" the molar proportions of A and T in this DNA are very differ- isolated by method (a) were used per assay. Exp. 4: same as ent. The same approach, namely compositional analysis of Exp. 3, but with the "fI RNA" isolated by method (b). Downloaded by guest on September 26, 2021 3838 Biochemistry: Karkas Proc. Nat. Acad. Sci. USA 70 (1973)

periment (Exp. 2) fi DNA *RNA hybrid was first exposed to Several of the viral polymerases, for instance, are reported to DNase and this "nicked" hybrid was used as template- exhibit RNase H activity (19-23); we have observed that primer. In two other experiments (Exps. 3 and 4), the RNA very small amounts of RNase H increase several-fold the strand of the fi DNA-RNA hybrid was first isolated by one template efficiency of natural hybrids for E. coli DNA poly- of the two techniques described (Methods) and then used as merase I (J. D. Karkas, unpublished observation). Within template; the primer was fi DNA separately fragmented the cell, it is very probable that polymerases and nucleases with DNase. Table 3 also includes, for comparison, the base act in concert as parts of large enzyme complexes involved in composition of fi DNA, "f1 RNA," and the DNA product DNA replication, repair, etc. But when dealing with isolated obtained in the DNA-directed synthesis, using intact DNA- enzymes, it is not possible to tell whether the presence of a RNA hybrid template. The results, and more specifically the nuclease in a polymerase preparation indicates a meaningful molar proportions of A and T, indicate that a DNA with a association in vivo of the two enzymes or is a mere artifact of base composition complementary to that of the "f1 RNA" is the isolation procedure. the main product of the reactions in which the latter is the Finally, it should be mentioned that reverse transcription template, because of the fragmentation of the DNA. by DNA polymerase I could be developed into a useful tool An additional proof of the complementarity of the newly for the in vitro synthesis of DNA strands complementary to synthesized DNA to the RNA template was provided by an biologically important RNA molecules provided that the examination of the susceptibility of the RNA to RNase H latter either carry a convenient "handle," like the poly(A) before and after its use in the DNA polymerase reaction. of some messenger , or can be supplemented with such. In Exp. 3 (Table 3) the amount of RNA solubilized by RNase The advantages of E. coli DNA polymerase in terms of avail- H after incubation with the DNA polymerase was higher ability, purity, stability, etc. over the viral enzymes exclu- than that solubilized at zero time, by an amount roughly sively used for this purpose until now are quite obvious. equal to that of the DNA synthesized by the polymerase. I thank Dr. Erwin Chargaff for his support and encourage- CONCLUDING REMARKS ment, and Dr. J. G. Stavrianopoulos for many helpful suggestions and discussions. I also acknowledge the excellent technical The conditions for reverse transcription by E. coli DNA assistance of Mr. Richard Liou. This work was supported by polymerase could probably be improved by a precise knowl- Research Grant CA-12210-13 from the National Institutes of edge of the sizes of the templates and primers and by a more Health, U.S. Public Health Service, and Grant NP-56P from the American Cancer Society. I am the recipient of an Irma T. systematic study of the parameters of hybridization and Hirschl Career Scientist Award. assay, especially with heteropolymeric RNA. What has be- come quite evident, however, from these experiments and 1. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1971) similar work in other laboratories [especially a report that Proc. Nat. Acad. Sci. USA 68, 2207-2211. in preparation is 2. Karkas, J. D., Stavrianopoulos, J. G. & Chargaff, E. (1972) appeared while this manuscript was (18)] Proc. Nat. Acad. Sci. USA 69, 398-402. the fact that under properly selected conditions DNA poly- 3. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) merase I of E. coli is capable of RNA-directed DNA synthe- Proc. Nat. Acad. Sci. USA 69, 1781-1785. sis in vitro, not only with homopolymer hybrids, but also with 4. Karkas, J. D. (1972) Proc. Nat. Acad. Sci. USA 69, 2288- all-ribonucleotide template-primers and even heteropolymeric 2291. 5. Stavrianopoulos, J. G., Karkas, J. D. & Chargaff, E. (1972) RNA. This is, therefore, not a unique property of the viral Proc. Nat. Acad. Sci. USA 69,2609-2613. RNA-directed DNA polymerases. Each DNA polymerase 6. Richardson, C. C., Schildkraut, C. L., Aposhian, H. V. & may have its own preferences for certain types of template- Kornberg, A. (1964) J. Biol. Chem. 239, 222-232. primers and certain sets of conditions, and such preferences 7. Jovin, T. M., Englund, P. T. & Bertsch, L. L. (1969) J. Biol. Chem. 244, 2996-3008. may be of great physiological importance; but the basic 8. Burgess, R. R. (1969) J. Biol. Chem. 244, 6160-6167. mechanisms underlying the reactions catalyzed in vitro by 9. Yoneda, M. & Bollum, F. J. (1965) J. Biol. Chem. 240, 3385- all known DNA polymerases do not seem to be different. 3391. This does not imply, of course, that the in vivo function of 10. Chang, L. M. S. & Bollum, F. J. (1971) J. Biol. Chem. 246, these enzymes is necessarily similar. Although the biological 909-916. 11. Kasai, K. & Grunberg-Manago, M. (1967) Eur. J. Biochem. roles of either DNA polymerase I or the viral enzymes are 1, 152-163. not clearly defined, it is easy to understand why reverse 12. Stavrianopoulos, J. G. & Chargaff, E. (1973) Proc. Nat. transcription might represent a vital step in the multiplica- Acad. Sci. USA 70, 1959-1963. tion of an RNA virus. A similar role for the E. coli enzyme is 13. Bollum, F. J. (1966) in Procedures in Nucleic Acid Research, eds. Cantoni, G. L. & Davies, D. R. (Harper & Row, New less easy to formulate; we do not know of any process in E. York and London), pp. 577-583. coli in which transcription of a single-stranded RNA into 14. Lee-Huang, S. & Cavalieri, L. F. (1963) Proc. Nat. Acad. DNA is required, although we cannot exclude the possibility Sci. USA 50, 1116-1122. that such a process may be discovered. However, one possible 15. Chamberlin, M. (1965) Fed. Proc. 24, 1446-1451. 16. Hurwitz, J. & Leis, J. P. (1972) J. Virol. 9, 116-129. use of RNA-directed DNA synthesis in normal cells would be 17. Shishido, K. & Ikeda, Y. (1971) J. Mol. Biol. 55, 287-291. in the repair of DNA * RNA hybrids which have been recently 18. Loeb, L. A., Tartof, K. D. & Travaglini, E. C. (1973) implicated in some important processes. Nature New Biol. 242, 66-69. A practical conclusion that can be drawn from the work 19. Molling, K., Bolognesi, D. P., Bauer, H., Busen, W., here is that of the effici- Plassmann, H. W. & Hausen, P. (1971) Nature New. Biol. described quantitative comparisons 234,240-243. encies of various template-primers, be they ribo- or deoxyribo- 20. Baltimore, D. & Smoler, D. F. (1972) J. Biol. Chem. 247, nucleotide, poly- or oligonucleotide, are not meaningful un- 7282-7287. less the lengths of both the template and the primer strands 21. Keller, W. & Crouch, R. (1972) Proc. Nat. Acad. Sci. USA are known to be the same. As for the comparison of various 69,3360-3364. 22. Grandgenett, D. P., Gerard, G. F. & Green, M. (1973) polymerase preparations with one and the same template- Proc. Nat. Acad. Sci. USA 70,230-234. primer, they are meaningful only if the enzymes being com- 23. Leis, J. P., Berkower, I. & Hurwitz, J. (1973) Proc. Nai. pared are highly purified and free of all possible nucleases. Acad. Sci. USA 70,466-470. Downloaded by guest on September 26, 2021