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Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part II, pp. 3834-3838, December 1973 Reverse Transcription by Escherichia coli DNA Polymerase 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 Enzymes. 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- nucleotide 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isolated from E. nuclease before being hybridized to the complementary coli by a modification (J. G. Stavrianopoulos, unpublished) strand, the template efficiency of the latter increases of a recent procedure (8).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 polymerases Polynucleotides. Poly(dA) and poly(dT) were prepared of certain RNA viruses. It was subsequently demonstrated in with terminal deoxynucleotidyl transferase, 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 phosphorylase, 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.

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