Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp. 965-969, April 1972 RNA Synthesis Initiates In Vitro Conversion of M13 DNA to Its Replicative Form (¢X174 DNA/DNA initiation/DNA replication/rifampicin) WILLIAM WICKNER, DOUGLAS BRUTLAG, RANDY SCHEKMAN, AND ARTHUR KORNBERG Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 Contributed by Arthur Kornberg, February 7, 1972 ABSTRACT Soluble enzyme fractions from unin- fected Escherichia coli convert M13 and ¢X174 viral single strands to their double-stranded replicative forms. Ri- fampicin, an inhibitor of RNA polymerase, blocks conver- sion of M13 single strands to the replicative forms in vivo and in vitro. However, rifampicin does not block synthesis of the replicative forms of ¢X174 either in vivo or in soluble extracts. The replicative form of M13 synthesized in vitro consists of a full-length, linear, complementary strand annealed to a viral strand. The conversion of single strands of M13 to the replicative form proceeds in two separate stages. The first stage requires enzymes, ribonucleoside triphosphates, and single-stranded DNA; the reaction is inhibited by rifampicin. The macromolecular product separated at this stage supports DNA synthesis with deoxyribonucleoside triphosphates and a fresh addition of enzymes; ribonucleoside triphosphates are not required in this second stage nor does rifampicin inhibit the reaction. We presume that in the first stage there is synthesis of a short RNA chain, which then primes the synthesis of a replicative form by a DNA polymerase. RNA polymerase (2), unlike the known DNA polymerases (8, 4, and Kornberg, T. and Gefter, M., personal communica- tion), can initiate as well as extend polynucleotide chains. It was this property of RNA polymerase that led us to inquire whether RNA polymerase might initiate the synthesis of a new DNA chain. We found (1) that the conversion of the single-stranded DNA circle (SS) of M13 phage into a double- stranded circle (replicative form, RF) in Escherichia coli was prevented by rifampicin, a specific inhibitor of RNA poly- merase (5). Inasmuch as this effect on RNA polymerase was not related to protein synthesis, a role for RNA polymerase in synthesizing a primer for initiating a DNA chain seemed likely. . We now report the conversion of M13 SS to RF with soluble enzymes extracted from E. colt and show with this system that a stage of RNA synthesis must precede DNA replication. Although chemical characterization of the synthetic product is incomplete, our results strengthen the suggestion (1) that RNA polymerase serves an essential role in priming DNA synthesis. RNA polymerase may have a similar role in other instances of replication in which a transcriptional event has been implicated. Examples are phage A (6), EB. coli (7), coli- cinogenic factor (Col E1) (8), and M13 RF (1). Silverstein and Billen (9); D.S. Ray, and L. McFadden and D. Denhardt (personal communications) have found that Abbreviations: SS, (phage) single-stranded circular DNA; RF, (phage) double-stranded circular replicative form of DNA; RF I, (phage) covalently closed RF; RF II, (phage) RF with a discontinuity in at least one strand. 965 conversion of ¢X174 SS to RF in E. coli is not inhibited by rifampicin. Their results indicate that RNA polymerase is not involved in formation of ¢X174 parental RF. Our enzyme studies show that there are distinctive mechanisms for con- version of the viral strands of M13 and ¢X174 to RF. MATERIALS AND METHODS Materials were from sources described previously (1). Ri- fampicin (Rifampin, Calbiochem) was used as before (1). [a«-#2?P ]dTTP was the generous gift of Dr. Robert H. Symons. M13 and ¢X174 phage were labeled with [*H]thymidine by the method of Forsheit and Ray (10) and Francke and Ray (11), respectively. SS were extracted by a minor modification of the method of Ray (12). Growth of Cells. E. coli H560 (F+, PolA1-, Endo I~) (13) was grown in Hershey broth at 37° with rotary shaking to 2 X 10 cells/ml and then chilled. The cells were collected and resuspended in 50 mM Tris-HCl (pH 7.5)-10% sucrose at 5-8 X 10” cells/ml and frozen in liquid nitrogen. No loss of activity (conversion of SS to RF) was seen in lysates after the cells were stored for up to 1 month. Cell Extract. Frozen cells (1 ml) were removed from liquid nitrogen, placed on ice for 20 min, then thawed in a 20° bath and immediately placed at 0°. Lysozyme (0.1 ml of 2 mg/ml in 0.25 M Tris-HCl, pH 7.5) and 25 ul of 4 M NaCl were added. After 45 min, the cells were warmed to 37° for 1 min, then chilled to 0°. The lysate was centrifuged for 15 min at 50,000 x g. The clear amber supernatant was made 10 mM in MgCh (by addition 10 yl of a 1 M solution) and stored at —20° (fraction A). Ammonium Sulfate Precipitation. 1 ml of fraction A was poured through a 0.6 X 1 cm DEAE cellulose column (DES2, H- Reeve Angel Inc., Clifton, N.J.) equilibrated with buffer A (50 mM Tris-HCl, pH 7.5, 10 mM MgCh, 10% sucrose, 0.1 M NaCl). Saturated ammonium sulfate (3 mI, neutralized with Tris base) was added at 0° to 1 ml of the material that had been treated with DEAE cellulose. After 30 min, the precipitate was collected and redissolved in 1 ml of buffer A. After dial- ysis near 0° for 4 hr against four 100-ml volumes of buffer A, the solution (fraction B) was stored at —20°. Assay of Conversion of SS to RF. The triphosphate mixture contained the four deoxyribonucleoside triphosphates, each at 150 uM, and ATP at 6 mM. In some experiments, dTTP or dGTP was labeled in the a position with "P at a specific activity. of 1 Ci/mmol. The four ribonucleoside triphosphates (1 mM each) were added to the mixture in some experiments. 966 Biochemistry: Wickner et al. T T T— RF () \ 4 ww wn T { uw | (0) + rifampicin Deoxyribonucleotide, p moles . x : : . T ! L i ! i 10 20 30 Fraction number Fie. 1. Conversion of M13 SS to RF in soluble extracts is blocked by rifampicin. Fraction A (100 ul) and unlabeled tri- phosphate mixture was used in (a) and (b); in (6), rifampicin (5 wl, 0.5 mg/ml) was added to fraction A 1 min before addition of #P-SS. The incubation mixture (120 yl) contained fraction A or B (100 ul), SS (273 pmol nucleotide in 10 ul of 10 mM Tris- HCl, pH 7.5), and triphosphate mixture (10 ul). Incubation was at 37°. The reaction was stopped by addition of sodium dodecyl sulfate (100 yl] of a 10% solution). After 15 min at 60°, 240 ul of water was added and the mixture was layered on a 5-ml sucrose gradient (5-20% in 10 mM Tris-HCl, pH 8, -1 mM EDTA-1 M NaCl) and centrifuged at 4° for 2.5 hr at 65,000 rpm in a Beckman SW 65 rotor; 0.1-ml fractions were col- lected. Scintillation fluid (5 ml of Triton X-100-toluene base) was added, and the samples were counted in a Nuclear Chicago scintillation counter. In some experiments, 10-x] aliquots of the reaction were added to 100 ul of 10% trichloroacetic acid; incorporation into the acid-insoluble fraction was measured (14). RESULTS Soluble-Enzyme Fraction Converts SS to RF. A soluble- enzyme fraction from gently lysed EF. colt (fraction A) cata- lyzed the conversion of SS to a form that co-sedimented with RF II (Fig. 1). Rifampicin, an inhibitor of RNA polymerase that blocks the conversion of SS to RF in intact cells at 200 ug/ml, prevented the in vitro reaction at 5 ug/ml (Fig. 1). RF synthesis similar to that shown in Fig. la was catalyzed by extracts of rifampicin-resistant E. coli in the presence of 5 ug/ml rifampicin. The RF product was characterized as RF II by velocity sedimentation in neutral and alkaline sucrose and by equilib- rium banding in alkaline CsCl as follows: (7) molar equivalence of the viral and newly synthesized DNA (Fig. 2a), (tt) Sedi- mentation of the new DNA as full-length linear strands and the viral strands mainly as circles (Fig. 26), and (vit) Banding of the new DNA at the density of M13 complementary strands (12) (Fig. 2c). On the basis of these findings, we con- clude that the in vitro product is RF II in which a newly synthesized, full-length linear complementary strand is an- nealed to a circular viral strand. Proc. Nat. Acad. Sct. USA 69 (1972) With fraction A, from which much of the cellular DNA has been removed, assay of the conversion of SS to RF could be measured directly by incorporation of a labeled nucleotide into an acid-insoluble material. Total nulceotide incorporation into the acid-insoluble material was identical to that identified in the RF region of sucrose velocity gradients (Table 1). Over 80% of the acid-insoluble nucleotide was RF. Conversion of M13 SS to RF as a function of time showed a requirement for added M13 DNA and inhibition by rifampicin (5 ug/ml) (Fig. 3). Conversion of ¢X174 SS to RF followed a time course simi- lar to that of M13 but was not inhibited by rifampicin (Fig. 3). RNA Synthesis Initiates Conversion of SS to RF. Enzyme treated with DEAE cellulose and precipitated with ammo- nium sulfate (fraction B) required the addition of ribonucleo- side triphosphates for conversion of M13 SS to RF (Fig. 4). A strong, but less complete, dependence was also seen in 1 T 7 T + Neutral sucrose 50 (G) Alkaline sucrose Oeoxyribonucizotide, pmoles 25-- 10 20 30 40 50 Fraction number Fie. 2. Characterization of RF synthesized in vitro. Triphos- phate mixture (40 ul with [*H)TTP, 2900 cpm/pmol), #P- labeled M13 SS (10 yl, 260 pmol, 1280 cpm/pmol), and fraction A (200 yl) were incubated at 37° for 5 min. Sodium dodecy] sul- fate was added (25 ul, 5%), and the RF was purified by sucrose gradient centrifugation and ethanol precipitation (1). 51 pmol of #2P-labeled viral DNA, V, and 53 pmol of *H-labeled com- plementary DNA, C, were recovered as RF. (a) Centrifugation of product DNA (5 pmol) for 4.5 hr at 4° through a neutral sucrose gradient [5-20% in 1.0 M NaCl, 1.00 mM EDTA, and 10 mM Tris-HCl (pH 8.0) in an SW56 rotor]. (b) Centrifugation of product DNA (5 pmol) for 4.5 br at 4° through an alkaline suc- rose gradient (5-20% in 0.8 M NaCl, 0.2 M NaOH, 1 mM EDTA in an SW 56 rotor). (c) Equilibrium alkaline CsCl sedimentation of product DNA (5 pmol) as described by Ray (12). Proc. Nat. Acad. Sci. USA 69 (1972) Taste 1. Assays of conversion of M18SS to RF* Deoxyribonucleotide incorporated Acid-insoluble SS added per RF fraction cell equivalent (pmol) (pmol) 1 4.7 4.8 5 20.2 23.3 * Isolation of RF compared to a direct assay of nucleotide incorporation into the acid-insoluble fraction. Fraction A (80 wl) was used for conversion of SS to RF. With 40 yl of the 100- wi reaction mixture, total acid-insoluble nucleotide was deter- mined; with another portion (40 ul), RF was isolated by sucrose velocity sedimentation. The values cited for RF are acid-in- soluble nucleotide in the RF region of a sucrose gradient. { 1 mg of protein = 3 X 10° cell equivalents. dialyzed or diluted extracts (data not shown). No single ribo- nucleoside triphosphate sufficed for efficient RF synthesis (Table 2); maximal activity was seen only when all four were present. . Does RNA synthesis precede DNA synthesis? Fraction B was incubated in a first-stage reaction with SS and the four ribonucleoside triphosphates in the absence of deoxyribo- nucleoside triphosphates. The mixture was filtered through Sephadex G-200 to remove the ribonucleoside triphosphates (less than 0.01% remained in the filtrate). In the second stage, the filtrate was treated with fresh fraction B and deoxyribo- nucleoside triphosphates. Formation of RF from SS required that the ribonucleoside triphosphates be present during the first, but not the second, stage (Table 3, compare Exps. 1 and 4). Rifampicin inhibited the first stage of the reaction but had a relatively small effect on the second (Table 3). DISCUSSION The conversion of M13 viral single strands (SS) to the double- stranded replicative forms (RF) upon infection of E. colt re- quires the action of RNA polymerase (1). This observation, based on the inhibitory effect of rifampicin, has now been Taste 2. Requirements for ribonucleoside triphosphates in the conversion of SS to RF Deoxyribonucleotide Ribonucleoside triphosphates added incorporated (pmol) ATP, GTP, UTP, CTP 7.8 ATP 0.4 GTP 0.7 UTP 0.0 CTP 0.0 None 0.1 The complete assay mixture contained fraction B (20 ul), deoxyribonucleoside triphosphates (2 ul containing dTTP, dCTP, dATP, and dGTP, each at 150 4M, with 0.1 mCi/ml of [a-*P]dTTP), and all four ribonucleoside triphosphates (1 yl each of 2.5 mM solutions of GTP, ATP, CTP, and UTP). After 15 min at 37°, RF synthesis was measured as incorporation of nucleotide into the acid-insoluble fraction. Priming of M13 DNA Synthesis 967 w oO o T a I - T + 9X 174 DNA +rifampicin +M13 DONA + rifampicin Deoxyribonucleotide incorporated, pmoles 3 T No jodded DNA ; : 2 4+ 6 & 10 Minutes Fig. 3. Conversion of SS to RF: rifampicin inhibits M13 but not ¢X174. Incubations contained fraction A, the four deoxyribo- nucleoside triphosphates (80 uM each), ATP (1 mM), buffer (Tris-HCl, pH 7.5, 4 mM), MgCl (5 mM), and one of the following: no DNA 0; M13 DNA + rifampicin, @; ¢X174 DNA: + rifampicin, A; ¢X174 DNA, A; or M 13 DNA, O. The added DNA was 100 pmol nucleotide/mg protein: 3 SS/cell equivalent. strengthened and extended by our studies with a soluble enzyme preparation from uninfected cells. / Soluble enzymes catalyze the formation of full-length linear strands on an M138 circular template (Fig. 2). The production of this replicative form (RF IT) can be separated into two stages (Table 3): an initial RNA synthesis followed + UTP, CTP, GTP » Ppmoles a T incorporated b T no addition Deoxyribonucicotide nN T ° L i ] 4 _ 8 12 Minutes Fie. 4. Conversion of M13 SS to RF requires ribonucleoside triphosphates. Fraction B (100 ul) was incubated with, O, or without, @, ribonucleoside triphosphates (100 nM each of GTP CTP, and UTP); ATP (1 mM) was present in both experiments. 968 Biochemistry: Wickner et al. TaBLe 3. Distinct stages in the conversion of SS to RF 1st Stage 2nd Stage (RNA synthesis) | (DNA synthesis) Deoxy- ribonu- UTP, UTP, cleotide Rifam- CTP, Rifam- CTP, incor- Experi- picin GTP picin GTP porated ment added added added added (pmol) 1 0 + 0 0 76 2 0 + + 0 66 3 + + 0 0 11 4 0 0 0 0 0 The incubation mixture (0.22 ml) contained fraction B (0.2 ml), SS (6 X 10" in 10 ul of 10 mM Tris-HCl, pH 7.5), ribonu- cleoside triphosphates (8 yl of a solution containing UTP, CTP, GTP, and ATP, each of 2.5 mM) and, where indicated, rifampicin (2 yl of a 0.5 mg/ml solution in 0.1 M Tris-HCl, pH 7.5). After 6 min at 37° (lst stage reaction), the solution was chilled and filtered through a column (0.6 X 7 cm) of Sephadex G-200 at 4°. Fractions were assayed at once because the first stage reaction product was unstable. An aliquot (20 ul) of the void-volume fraction was mixed with [a-"P]dTTP triphosphate mixture (5 ul), fraction B (20 ul) and, where indicated, rifampicin (1 ul of 0.25 mg/ml). The solution was incubated at 37° for 15 min (2nd stage reaction) and precipitated with acid. In control experi- ments, there was no detectable contamination (<0.01%) of void- volume fractions with ribonucleoside triphosphates. by DNA synthesis. The first requires the four ribonucleoside triphosphates and is inhibited by rifampicin. A macromolecu- lar product separated after this stage supports the synthesis of the complementary DNA strand in the absence of ribonu- cleoside triphosphates and in the presence of rifampicin. We presume that a short segment of RNA synthesized in the first stage of RF formation serves as a primer for the extensive DNA synthesis in the second. However, our at- tempts to determine the size and composition of this initiating RNA segment have been frustrated by the synthesis of non- specific RNA by these relatively crude enzyme fractions. Unlike the DNA synthesis, this RNA synthesis (70 pmol/mg protein) is independent of the presence of M13 DNA; the RNA resists pancreatic RNase (25% persists) and contami- nates the RF II product in density gradient separation. Fur- ther purification of the enzymes involved is required to clar- ify this problem. Even though the RNA priming segment has not been iden- tified, its existence is supported by evidence in addition to that already cited or reported here. The isolated RF II contains a 5’ —» 3’ phosphodiester link of a deoxyribonucleotide to a ribonucleotide. Alkaline hydrolysis of the RF II synthesized in the presence of four [a-??P]deoxyribonucleoside triphos- phates, yielded *?P-labeled ribonucleotide (1.47 mol/mol RF II) of which 84% was a mixture of 2’- and 3’/-rAMP (Brutlag and Kornberg, unpublished data). A Y Z@ x ou bon }a LH -~---) Sp, | pa spa spe - --- alkali cleavages Another line of evidence suggesting the role of an RNA primer is the presence at the 5’ end of the synthesized DNA Proc. Nat. Acad. Sct. USA 69 (1972) of a nucleotide structure, conceivably a 5’-ribonucleoside tri- phosphate derivative, which cannot be sealed by ligase. At- tempts to convert the RF IT to RFI by ligase, in the presence of DNA polymerase and deoxyribonucleoside triphosphates, succeeded with FE. coli DNA polymerase I but not with T4 DNA polymerase. The £. coli enzyme, whose 5’ — 3’ exo- nuclease function can excise nucleotides terminating in 5/- triphosphate and mismatched regions at the 5’ end, converted 35% of the RF II to RF I, whereas the T4 enzyme, which lacks this excision function, was inert in this regard (Brutlag and Kornberg, unpublished data). These data may also ex- plain the failure of these extracts that are deficient in DNA polymerase I to seal covalently the RF synthesized in vittro. Finally, model experiments with pure RNA and DNA poly- merases show that the synthesis of a small piece of RNA primes the use of an otherwise inactive M13 DNA template (Bertsch and Kornberg, unpublished experiments). The ca- pacity of DNA polymerases to extend a variety of oligoribo- nucleotides annealed to templates has been thoroughly ex- plored by Wells and coworkers (15). Is RNA synthesis involved in the initiation of M13 DNA synthesis in the cell as it is in these extracts? The identical behavior of rifampicin in vivo (1) and in miro suggests that it is. Even more persuasive is the evidence showing the specificity of the enzymes catalyzing M13 and ¢X174 replication. In the same bacterial strain (H560), rifampicin inhibits conver- sion of M1385 to RF in vivo but not that of the rather similar ¢X174§8; extracts of this strain show the very same behavior (Fig. 3). Furthermore, certain £. coli mutants (dna B), which fail to synthesize host DNA at an elevated temperature and which support M13 but not ¢X174 parental RF formation (16, and Ray, personal communication) at the restrictive temperature, show an analogous behavior with enzyme frac- tions from this mutant (Schekman and Kornberg, unpub- lished experiments). Purification of the dna B gene product should reveal whether initiation of ¢X174 8S replication in- volves a rifampicin-resistant RNA synthesis or some other mechanism. There are strong indications that RNA polymerase action is also involved in the replication of double-stranded DNA. Multiplication of M13 RF is immediately and profoundly inhibited by rifampicin (1); so is the replication of the RF-like colicinogenic factor Col Ei (8). Furthermore, Blair et al. (8) have found that alkaline or RNase digestion introduces breaks in the supercoiled Col F1, suggesting that a segment of RNA is present in the supercoiled DNA. Dove has implicated a transcriptional event in the initiation of phage \ DNA replica- tion (6) and Lark has observed rifampicin sensitivity of the start of E. coli chromosome replication that he also attributes to an RNA synthetic action (7). Although the nature of the RNA polymerase action is uncertain in each of these instances, a primer function for DNA initiation appears to us as the most attractive explanation. This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. W. 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