Purification of Ribonuclease H As a Factor Required for Initiation of in Vitro Colel DNA Replication

Volume 10 Number 19 1982 Nucleic Acids Research

Purification of ribonuclease H as a factor required for initiation of in vitro ColEl DNA replication

Tateo Itoh and Jun-ichi Tomizawa

Laboratory of Molecular Biology, National Institute of Arthritis, Diabetes, and Digestive and Kid- ney Diseases, National Institutes of Health, Bethesda, MD 20205, USA

Received 2 July 1982; Revised and Accepted 1 September 1982

ABSTRACT Escherichia coli ribonuclease H was purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro ColEl DNA replication from the unique origin by RNA polymerase and DNA polymerase I. Both ribonuclease H activity and stimulating activity for ColEl DNA synthesis comigrate with the single protein band in gel electrophoresis. These two activities coincide throughout the process of purification. Some DNA synthesis takes place on covalently closed-circular DNA molecules other than ColEl DNA with the three purified enzymes. This DNA synthesis is suppressed by an Esherichia coli single-strand DNA binding protein and/or a high concentration of ribonuclease H. Negative superhelicity of template DNA is required for efficient primer formation. No evidence that supports involvement of ribonuclease III in iniilation of ColEl DNA replication or its regulation was found.

INTRODUCTION Colicin El plasmid (ColEl) can be replicated in vitro by a soluble enzyme systeml. This replication starts at a unique site on the genome2 and does not require plasmid-coded proteins3. We have previously shown that Escherichia coli ribonuclease H (RNase H) is required for initiation of in vitro ColEl DNA replication by RNA polymerase and DNA polymerase I4. In this paper we describe, in detail, the procedure of purification of RNase H as a factor required for initiation of in vitro ColEl DNA replication and some properties of DNA synthesis mediated by RNA polymerase, RNase H and DNA polymerase I. The mechanisms of primer formation by RNA polymerase and RNase H5 and of its regulation by a plasmid-specified small RNA (RNA I) have been described6,7.

MATERIALS AND METHODS Bacteri and plsmids.E. coli K12 NT5258 grown in H broth to the end of the logarithmic phase was used as a source of RNase H. E. coli strains AB301-1059, N207710 and BL10711 possess the same rnclO5 mutation in different genetic backgrounds. Plasmids used were ColEll and its small derivatives pNT112 and pNT75.

C IRL Press Umited, Oxford, England. 5949 0305-1048/82/1019-5949$ 2.00/0 Nucleic Acids Research

Nucleic Acids. Supercoiled molecules of plasmid DNA were purified after extensive treatment with RNase A as described4. Relaxed closed circular molecules of pNT7 DNA were prepared by treating supercoiled molecules with the superhelical DNA relaxing enzyme13 from HeLa cell nuclei which was a gift of N. Nossal. Xdvl DNA and *X174 RFI DNA were purchased from Boehringer Mannheim QmbH and Bethesda Research Laboratory, respectively. Phage fl RFI DNA and SV40 Form I DNA were gifts of G. Selzer and N. Salzman, respectively. Enzymes. E. coli single-stranded DNA binding protein (SSB) and a sample of RNase H were gifts of S. Wickner and J. Hurwitz, respectively. The holo- enzyme of RNA polymerase and DNA polymerase I (provided by A. Kornberg) were as described5. RNase III was prepared as describedl4. Buffers. Buffer I: 50 mM Tris-HCl pH 7.5/0.1 M NaCl/10% sucrose. Buffer II: 50 mM Tris-HCl pH 7.5/0.1 M NaCl/l mM dithiothreitol (DTT)/l mM EDTA/20% glycerol. Buffer III: 20 mM Tris-HCl pH 8.9/3 mM DTT/l mM EDTA/10% glycerol. Buffer IV: 20 mM Tris-HCl pH 7.5/3 mM DTT/l mM EDTA/10% glycerol. Buffer V: 50 mM Tris-HCl pH 7.5/0.3 M NaCl/l mM DTT/1 mM EDTA/20% glycerol. Buffer VI: 50 mM Tris-HCl pH 7.5/0.1 M NaCl/l mM DTT/l mM EDTA/50% glycerol. Other Materials. DNA-agarose prepared as described15 was a gift of R. E. Bird. All other materials were obtained from commercial sources. RNase H Assay. The standard reaction mixture (50 il) for RNase H assay'6 contained 40 mM Tris-HCl pH 7.8/4 mM MgCl2/1 mM DTT/22 PM [3H]poly A (15,000 cpm/nmol AMP)/25 jiM poly dT/30 pg/ml of bovine serum albumin/4% glycerol and an appropriate amount of RNase H. RNase III Assay. RNase III activity was measured as described using poly(r[14C]A-U) (6000 cpm/nmol) as substrate 17. Transcription and DNA Synthesis. Conditions for transcription of plasmid DNA have been described5-7. The standard reaction mixture (30 jil) for DNA synthesis4 contained 23 mM potassium phosphate buffer pH 7.4/7 mM Tris- HCl pH 7.8/8 mM MgC12/60 mM KC1/2 mM DTT/10% glycerol/2 mM spermidine/400 jiM ATP/200 PM each of other NTPs/25 jiM dNTPs including [a(-32P]dTT (500 to 2000 cpm/pmol)/100 jg/ml bovine serum albumin/5 units/ml RNA polymerase/25 units/ml DNA polymerase I/1.5 units/ml RNase H, or an appropriate amount of an enzyme fraction/10 jg/ml of DNA, usually ColEl DNA. Supercoiled molecules were used. Assays of Enzymatic Impurities. DNase: To assay for endonuclease, 0.7 jig of covalently closed circular ColEl DNA was incubated with 0.5 unit of RNase H in the RNase H assay mixture for 60 min at 30°C and then analyzed by 0.7% agarose gel electrophoresis. For assay of exonuclease, 0.01 pg [3H]thymidine labeled E. coli DNA (6 x 106 cpm/jg),either native or denatured, was incubated with 10 units of RNase H in the RNase H assay mixture for 60

5950 Nucleic Acids Research min at 37C and the formation of acid-soluble radioactivity was measured. RNase: Phage Xb2 transcripts that were used for assay of RNases were prepared by incubating Xb2 DNA (45 ig/ml) with RNA polymerase (15 units/ml) in a reaction mixture containing 20 mM Tris-HCl pH 7.9/5 mM MgC12/100 mM KC1/0.1 mM DTT/0.1 mM EDTA/150 pM NTPs including [a-32P]ATP (1,200 cpm/pmol) for 3 min at 37'C. RNA was purified by phenol treatment and concentrated by alcohol precipitation. [32P]Xb2 transcripts (0.02 pg: 2 x 104 cpm) were incubated with 5 units of RNase H in the RNase H assay mixture for 60 mmn at 370C. The formation of acid-soluble radioactivity was used as a test for exonuclease and large amounts of endonuclease. The products were also analyzed by polyacrylamide/ urea gel electrophoresis to provide a more sensitive assay for any endonucle- ases including RNase III5. RNase III will give rise to disappearance of long XPL promoted transcripts and appearance of defined cleavage products 5 Other Methods. NaDodSO4/polyacrylamide (15%) gel electrophoresis of proteins was as describedl8. Protein was determined as describedl9,20 using bovine serum albumin as the standard.

RESULTS Purification of RNase H. For purification of RNase H to near-homogeneity, it was necessary to modify the published procedure21. We found that the previous method invariably yielded products that contained RNase III activity detectable by specific cleavage of XPL-promoted transcripts, and several other contaminating proteins. The rationale of the modification is as follows. When the previous method of DEAE cellulose chromatography at pH 7.5 was applied, RNase H activity, different from the "RNase H" activity of DNA polymerase I and exonuclease III was found both in the flow-through fraction and in the fraction that eluted at around 0.05 M NaCl as describedl6. The ratio of activities in these fractions varied from experiment to experiment. We inferred that this was due to a very slight affinity of RNase H for DEAE cellulose at pH 7.5. We therefore performed the chromatography at two different pHs. With Buffer III (pH 8.9), all RNase H activity was bound to DEAE- cellulose. With Buffer IV (pH 7.5), the enzyme eluted after unadsorbed material without application of a salt gradient. Successive use of these two conditions of DEAE cellulose chromatography removed RNase III and some contaminating proteins. A protein(s) that was eliminated by these procedures migrated at the same position as RNase H in gel electrophoresis. This contaminating protein was not retained at all by DEAE cellulose at pH 7.5. If not separated at this step, it was difficult to remove completely by the succeeding procedure. Some details of the method of purification are described

5951 Nucleic Acids Research below and in Table 1. All the operations were performed at around 4°C and centrifugation was at 20,000 g for 90 min. Chromatographic columns were always equilibrated with the starting buffer and the samples applied were dialyzed against the same buffer before loading. Cells of E. coli NT525 (206 g) in 600 ml of Buffer I were disrupted by passage through a Manton-Gaulin homogenizer (13,000 psi) and the debris were removed by centrifugation. To the supernatant (Fraction I, 720 ml), 226 g of ammonium sulfate was added and the precipitate was dissolved in 90 ml of Buff- er II. The solution (Fraction II, 135 ml) was applied onto a Bio-Gel A-5m column (5 x 80 cm) which was then washed with 2 1 of the buffer. RNase H activity eluted after about one column volume. Fractions (236 ml) containing RNase H activity, except those eluted earlier and contained DNA polymerase I activity, were pooled and 62 g of ammonium sulfate was added. To the supernatant obtained by centrifugation, 60 g of ammonium sulfate was added. The precipitate was dissolved in 7 ml of Buffer III. The dialyzed solution (Fraction III, 17 ml) was applied to a DEAE cellulose (Whatman DE52) column (7.5 x 21 cm). The column was washed with Buffer III (500 ml) and then developed with a 0 to 1 M linear gradient of NaCl in the same buffer (total 2 1). The RNase H activity eluted at around 0.06 M NaCl. Active fractions (120 ml) were pooled and 70 g of ammonium sulfate was added. The precipitate was dissolved in 1.2 ml of Buffer IV. The dialyzed solution (Fraction IV, 2.4 ml) was applied to a DEAE cellulose column (2.3 x 40 cm) and the column was washed with Buffer IV (200 ml). The RNase H activity eluted from the column well after the bulk of unadsorbed proteins (Fig. 1A). The active fractions (Fraction V, 20 ml) were pooled and applied to a phosphocellulose (Whatman Pll) column (0.9 x 12 cm). The column was washed with Buffer IV (50 ml) and developed with a 0 to 1 M linear gradient of NaCl in the same buffer (total 500 ml). The fractions (10 ml) with RNase H activity, which eluted at around 0.34 M NaCl (Fig. 1B), were pooled, diluted two-fold with Buffer IV and applied to a phosphocellulose column (0.5 x 2.5 cm). After washing with Buffer IV (5 ml), the RNase H activity was eluted with 0.5 M NaCl in the same buffer (5 ml). The active fractions (Fraction VI, 1.2 ml) were pooled and concentrated by dialysis against a dry powder of Sephadex G-50 (fine). Half of the resultant solution (0.2 ml) was applied to a Sephadex G-50 (superfine) column (0.7 x 60 cm) and the column was washed with Buffer V (20 ml). The active fractions (1.3 ml) (Fig. 1C) were pooled and concentrated by dialysis against a dry powder of Sephadex G-50 (fine). After dialysis against Buffer VI the material was used as the final preparation (Fraction VII, 0.65 ml). Fraction VII has a specific activity of 470,000 units/mg of protein. The

5952 Nucleic Acids Research

0) 4-4 0) 0 H 11-' 0 0 r-H 00 Ai 0 4H 0 4-i --4.4 5- r-H '-4 P-H r- oO.40)0)0 0) 0)c)00) -W -H v 10 O c) "-I 0 a- ,0)0) 4-4C. 0. 54 0)0 54 0) 0) 0) - 4 0 O O 0 '0 .-4 4-i .,4 CIP O O) 0 co 0 -H -H ci, '0) co > Vi n JJS (VI) -H I 0) 4i C) 9 to C3 4O 0 VHO--0)N C) 0) C co coz 0 H 00 0 0 00 0 4J " 0)0 0) 0) CO Oq ON O 0 _I =,1 0 :C c--O u-i 0 0) o - c 0) 0)-40)4 0)0) co 0) -Hi 0) 0 w 0 4-4co '0) C 4-4 0) 0) 0 O0 0 4J 0 01 00 4-H -H co co V4-0 4i 0 CO 0) -H r-4 0)0 4 4-i zZ4-i" 0 -Hi 0) Lf)s 0) 'u8' 0)- 0 0 0 O 0 0 10 0 0 0 0 0 O -H 0) 0 0 0 0 %D 0 Oi c C 0' 0 V0 4 ci, r-H ON ec 4-i 0)000 0) W 4o34 _ r-H la0) O- co<0 o -C CV4 c-oI 0)0 0) IV > 0 0 -H r- 0) 0 0) 0) 0)C 0)0 0 0 C--s 0 0 0 0 0 0 10 0 uV 0)0) 0) 5- I 0-H co0 -H X P)44v0O0

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5953 Nucleic Acids Research

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5954 Nucleic Acids Research preparation apparently contains only one protein, as judged by gel electrophoresis (Fig. 2). When Fraction VI was chromatographed on DNA-agarose or hydroxylapatite (Bio-Gel HTP), the RNase H activity eluted in a single peak. In either case the active fractions contained the same protein found in Fraction VII when analyzed by gel electrophoresis (data not shown). Therefore most of the protein in Fraction VII is likely to be the enzyme. When covalently closed circular ColEl DNA was incubated with the Fraction VII, no decrease of the substrate DNA or increase of open circular form was

VIl VI V IV III 11 I

F-_

Figure 2. NaDodSO4/polyacrylamide gel electrophoresis of fractions at each step of purification of RNase H. The amount of proteins applied were as follows: Fraction I, 95 jg: Fraction II, 85 jig; Fraction lll, 90 pg; Fraction IV, 90 jig; Fraction V, 7 jg; Fraction VI, 1 pg; Fraction VII, 3 pig. Bovine serum albumin (66,300 daltons), a subunit of E. coli RNA polymerase (36,500 daltons), soy bean trypsin inhibitor (21,500 daltons), and a subunit of 0-lactoglobulin (18,400 daltons) were used as molecular weight markers and are indicated by arrows on the left of the figure from top to bottom.

5955 Nucleic Acids Research detected. No detectable acid-solubilization of E. coli [3H]DNA was observed. Neither the size nor number of [32p]Xb2 transcripts was changed after incubation with the fraction. The molecular weight of RNase H was approximately 21,000 in the denatured state (Fig. 2) as well as in the native state as determined by sucrose density gradient centrifugation or Sephadex G-75 column chromatography (not shown). Identification of RNase H as a Factor Required for Initiation of in vitro ColEl DNA Replication. Fractions containing RNase H at each step of purification from Steps IV to VII were assayed for their ability to stimulate ColEl DNA synthesis by RNA polymerase and DNA polymerase I. We found that this activity copurified with the RNase H activity (Table 1, Fig. 1). Furthermore when Fraction VII was analyzed by gel electrophoresis and activities of protein eluted from the gel slices were measured, both activities migrated at the same position as the only protein band detected when the neighboring lane in the gel was stained (Fig. 3). The identity was further supported by

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10 20 30 40 50 60 70- 80 FRACTION NUMBER

Figure 3. Comigration of RNase H activity (-) and ColEl DNA synthesis stimulating activity (o) on NaDodS04fpolyacrylamide gel electrophoresis. Fraction VII (3 jg) was applied in the parallel lane on the gel shown in Figure 2. After electrophoresis the gel strip corresponding to the lane was cut out and soaked in Buffer V for 30 min at 40C. Then the strip was cut into 1-mm slices, which were incubated in 0.1 ml of Buffer V containing 200 pg/ml of bovine serum albumin for 10 hr at 40C. Slices 1 to 45 and 55 to 80 were combined by every 5 slices. Two activities in each fraction or combined fractions were determined. Electrophoresis was from left to right. The thick arrow indicates the position of the only protein band stained in the parallel lane. The thin arrows indicate the positions of cytochrome c (12,400 daltons), soy been trypsin inhibitor and DNase I (31,000 daltons) from right to left.

5956 Nucleic Acids Research the fact that the two activities were destroyed at a similar rate by treatment with either 10 mM N-ethylmaleimide (a half life of about 5 min in Buffer V at 30°C) or with heat (a half life of about 7 min in Buffer V at 75°C). Characterization of ColEl DNA Synthesis by Three Purified Enzymes. The kinetics of DNA synthesis by RNA polymerase and DNA polymerase I with or without RNase H, along with rifampicin sensitivity of the reaction, are shown in Figure 4. DNA synthesis without RNase H is considerably lower than that reported previously4 because of the greater purity of the enzymes (particularly DNA polymerase I) used here. The effects of varying the concentrations of RNase H, RNA polymerase and DNA polymerase I are shown in Figure 5A, B and C, respectively. The effect on ColEl DNA synthesis of varying the concentration of several components of the reaction mixture was examined (data not shown). We found rather broad ranges in the allowable concentrations of KCl and MgCl2 with op- timal synthesis around 100 mM and 8 mM, respectively. The optimum concentration of the four dNTPs was 25 liM each. Addition of spermidine (up to 2 MM) had little effect although a slight stimulation at around 0.5 mM was seen. DNA synthesis increased as the concentration of the four NTPs was increased to 40 jM each and decreased very gradually at higher NTP concentrations. In contrast, for DNA synthesis in a cell extract, the optimum concentration is about 200 jiM eachl. This requirement of high concentration of NTPs is, at least in part, due to degradation of NTPs during incubation with a cell extract. The location of initiation sites of DNA synthesis on pNTl DNA by the three enzymes was examined. As was found previously with a mixture of less pure

Figure 4. Kinetics of ColEl DNA synthesis by RNA polymerase and DNA polymerase I in the presence (o,e) or absence (A,A) of RNase H. 1.5/.5/oDNA synthesis with (e,A) or without (o,A) rifampicin (20 jg/ml) was determined. x 1.0 0

5957 Nucleic Acids Research

t z cc

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c-oL X VVd3 c-OL X VYd C0

5958 Nucleic Acids Research

enzymes, the majority of the products were found to have initiated exactly at the same positions in the origin region of ColEl as found in cell extracts4 (data not shown). No DNA fragments initiated at the unique origin were found among the DNA products synthesized without RNase H (data not shown). Effect of Structures of ColEl DNA on DNA Synthesis by Three Purified Enzymes. ColEl DNA synthesis by three purified enzymes was not observed when relaxed closed circular molecules were used as template4. We then examined the effect of the structure of template DNA on transcription of the plasmid pNT7 (Fig. 6A). Relaxed closed circular molecules of the plasmid were poor templates for synthesis of primer transcripts as for synthesis of RNA I22, but synthesis of a-lactamase mRNA was hardly affected by the structure of the template DNA. Next, effect of structures of template DNA on the efficiency of primer formation was examined. Assuming that the low efficiency

A B Figure 6. Effect of structure of pNT7 DNA on transcription (A) and on primer formation (B). (A) RNA synthesis was carried out as described4 on supercoiled (lane 1), or relaxed closed circular (lane 2) molecules of pNT7 DNA. Roman VI * numerals indicate the position of the corresponding RNA species5. Species VI V , is a-lactamase transcripts and species VIII is the primer RNA. (B) Transcription from the primer promoter on supercoiled pNT7 DNA was Vill * Vll es initiated by using a dinucleotide, UpU7. Prior to subsequent elongation step of the transcripts, the template DNA was cut by the restrction endonuclease EcoRI and transcripts labeled by [ca-32P] AMP were analyzed by electrophoresis in 3% polyacrylamide/urea gel as described5 (lane 2). Transcripts formed on the intact template DNA were also examined (lane 1).Species V is the primer tran- script terminated just before entering the 8-lactamase gene5.The arrow indicates the position of the run-off transcripts. Small transcripts in lane 2 are products of pausing of transcription.

5959 Nucleic Acids Research of synthesis on the relaxed template was due to poor initiation of transcription, transcription was initiated selectively from the primer promoter on negatively supercoiled pNT7 DNA using uridylyl (3' + 5') uridine7. Prior to subsequent elongation step of primer transcripts in the presence of RNase H, the template DNA was cut with the restriction enzyme EcoRI at the site 153 base pairs downstream from the origin. The results in Fig. 6B show that among the RNA molecules that were transcribed through the origin, nuch fewer were cleaved by RNase H when the template DNA was cut by EcoRl than when it was intact. When a similar experiment was performed without RNase H, the amount of primer precursor was reduced by cutting the template DNA with EcoRl (data not shown). These results indicate that efficiency of primer formation depends on the structure of the template DNA, but the dependence is incomplete. A large fraction of circular ColEl DNA molecules prepared from bacteria incubated with chloramphenicol ("amplified") contains ribonucleotides23 in one or both strands at more or less random positions24. In the standard conditions, these were as good templates for transcription and DNA synthesis as those which did not contain ribonucleotides (data not shown). RNA polymerase- independent DNA synthesis in the presence of RNase H (1 unit/ml) was similar for both kinds of templates. It appears that cleavage of the ribonucleotide sequences in DNA is a slow process and DNA synthesis resulting from nick translation by DNA polymerase I is inefficient compared to RNA polymerase/ RNaseH-dependent DNA synthesis. Template Specificity of DNA Synthesis by Three Purified Enzymes. Effect of RNase H. The in vitro DNA synthesis in a cell extractl is specific to ColEl as template and other DNAs including Xdvl, fl RFI, fXl74 RFI and SV40 Form I DNAs have no template activity (data not shown). While we were searching for a factor involved in initiation of ColEl DNA synthesis from the unique origin, we found that at least two protein fractions could distinguish ColEl DNA from other DNA species. One of them was a fraction from the second DEAE cellulose column containing a stimulating factor for ColEl DNA replication which was later identified as RNase H. This fraction could stimulate DNA synthesis by RNA polymerase and DNA polymerase I on various DNA species at around a concentration just enough for maximum stimulation of ColEl DNA synthesis (not shown). When present in excess, it inhibited DNA synthesis on template DNAs other than ColEl DNA. Therefore we tested if these effects were due to RNase H by using the Fraction VII enzyme. Xdvl and fl RFI DNAs showed a significant template activity in the DNA synthesizing system of three purified enzynes

5960 Nucleic Acids Research under the standard condition with 1 unit/ml of RNase H (Fig. 7A). However at high concentrations of RNase H, DNA synthesis on fl RFI and \dvl DNAs was severely inhibited, while that on ColEl DNA was affected only a little. 4XL74 RFI and SV40 Form I DNAs showed very low template activities and there was little effect of RNase H on DNA synthesis except for a slight stimnulation around 1 unit/ml of RNase H. Therefore, when the concentration of RNase H is high, DNA synthesis by the three purified enzymes is specific to ^co!'E. Effect of single-stranded DNA binding protein (SSB). The other fraction which could distinguish ColEl DNA from other DNA species adsorbed to DEAE cellulose and phosphocellulose (data not shown). Anong those well-character- ized proteins which participate in DNA metabolism and adsorb to DEAE cellulose and phosphocellulose is E. coli SSB, which is known to be required along with RNase H and two other proteins for discrimination between fd viral DNA and *X174 viral DNA as template in a reconstituted DNA synthesizing system21. The SSB inhibited ColEl DNA synthesis by about 20% at the highest concentration (2.2 jg/ml) of SSB in conformation of our previous results3 (Fig. 7B). On the other hand, fl RFI DNA synthesis with or without RNase H was severely inhibited by SSB (Fig. 7B). Similar severe inhibition was observed when 4X174 RFI, Xdvl and and SV40 Form I DNAs were used (data not shown). With 2.2 jig/ml of SSB, there was no significant DNA synthesis or its stimulation by RNase H on DNAs other than ColEl. Therefore, in the presence of SSB, DNA synthesis with three purified enzymes is specific to ColEl DNA even at a low concentration of RNase H (Fig. 7C). The sizes of DNA fragments synthesized on ColEl DNA were heterogeneous as revealed by gel electrophoresis (data not shown). DNA sy!ithesit terminated at many unique sites that were different depending upon the presence or absence of SSB. The length of the longest fragments formed i, the presence of SSB depended on the contour length of the template DNA and was always about 7% of it. When DNA gyrase was included the fragments further elongated4. When SSB was not added, some DNA fragments had a structure which was formed due to template-switching by DNA polymerase I. Inability of RNase III to replace RNase H. The possibility of involvement of RNase III in ColEl DNA replication in vivo25, or in initiation of ColEl DNA synthesis in vitro26 has been suggested. We have, however, found that cell extracts prepared from three RNase III-deficient strains9-11 can support normal initiation of ColEl DNA synthesis to form early replicative intermediates7 (data not shown). Mbreover, in vivo ColEl DNA replication has been shown to be independent of the presence of active RNase III7,27.

5961 Nucleic Acids Research

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5962 Nucleic Acids Research

When purified RNase III (0.3 to 300 units/ml) was added to the reaction containing RNA polymerase and DNA polymerase 1, the enzyme could not replace RNase H in stimulation of ColEl DNA synthesis. Moreover, additon of RNase III (0.3 to 300 units/ml) to the reaction containing the three eizyuaes showed no effect on ColEl DNA synthesis (data not shown).

DISCUSSION E. coli RNase H has been purified to near-homogeneity and identified as the only additional factor required for initiation of in vitro ColEl DNA replication by RNA polymerase and DNA polymerase I. The molecular weight of RNase H was determined to be about 21,000 either in the denatured or the native condition. The molecular weight in the native state was previously reported to be about 21,0004,14,28, 35,00029 or 40,00030. ColEl DNA synthesis by the three purified enzymes was observed only on negatively supercoiled template DNA. Moreover, negative superhelicity of the template DNA is required for efficient initiation of transcription from the primer promotor and for efficient formation of the primer as well. Elonga- tion of DNA chains is also facilitated by negative superhelicity of the template DNA4. We reported previously that formation of the early replicative intermediates from exogenously supplied supercoiled ColEl DNA in an cell extract is blocked by inhibitors of DNA gyrase8. Inefficient initiation of DNA synthesis in cell extract due to relaxation of the template DNA in the absence of compensating DNA gyrase activity provides an explanation4. In the presence of RNase H at a high concentration and/or SSB, DNA synthesis by RNA polymerase, RNase H and DNA polymerase I is specific to a ColEl DNA template, just like that in a cell extract. Since the concentration of RNase H in cell extracts is estimated to be 100 units/ml or more (data not shown), RNase H itself may be one of the factors which endows specificity for template DNA molecules by removing RNA molecules that are synthesized by RNA polymerase in the reaction, are left attached to the template DNA and can serve as non-specific primers for DNA synthesis. Stimulation of DNA synthesis on templates other than ColEl by a low concentration of RNase H may be due either to removal of RNA molecules from the templates to facilitate elongation of the DNA chain or creation of new 3'-hydroxyl priming ends for DNA synthesis. It has been reported that SSB has only a small effect on DNA synthesis by DNA polymerase I, and that it severely inhibits transcription by RNA polymerase on single-stranded DNA but only slightly on duplex DNA31. The role of SSB in our system may be to inhibit synthesis of RNA molecules which can serve as non-

5963 Nucleic Acids Research specific primers, or to displace such RNA molecules from template DNA. It has been reported that a protein fraction containing RNase H has a discriminatory function in the selective initiation of ColEl-type plasmid DNA replication32. A mutant of E. coli with an altered level of RNase H (about 30% of the parental level) has been described, and stable maintenance of a derivative of ColEl in this strain has been shown 33. It is quite probable, that the re- sidual RNase H activity in this mutant is sufficient for the stable maintenance of ColEl. We do not know if the enzyme is involved in ColEl DNA replication in vivo. However, the fact that these three enzymes can start ColEl DNA synthesis from the same unique origin as in vivo replication34 strongly sug- gests that this is the case. Finally we comment on another role of RNase H in ColEl DNA replication: namely, elimination of the RNA primer from the DNA products. When ColEl DNA synthesis was carried out with the three purified enzymes in the presence of all four NTPs and dNTPs, the covalently joined RNA primer was removed from the DNA products5. It is not known whether RNase H is solely responsible for elimination of all the ribonucleotides or whether the 5' + 3' exonuclease activity of DNA polymerase I also participates in the process. Elimination of the RNA primer from a DNA product by RNase H may be a genaral :leclaritism that operates in DNA replication.

ACKNOWLEDGMENT We are grateful to D. Apirion, R. E. Bird, R. Crouch, J. Hurwitz, A. Kornberg, N. Nossal, N. Salzman, G. Selzer, F. W. Studier and S. Wickner for providing us some materials used in this work. We thank D. Rogerson for growing a large quantity of bacterial cells for -is. We acknowledge the critical reading of the manuscript by M. Gellert and G. Selzer, and help in preparation of the manuscript by S. Butler.

*Present address: Osaka University, Faculty of Science, Toyonaka, Osaka 560, Japan.

REFERENCES 1. Sakakibara, Y. and Tomizawa, J. (1974) Proc. Natl. Acad. Sci. USA 71, 802-806. 2. Tomizawa, J., Olinori, H. and Bird, R. (1977) Proc. Natl. Acad. Sci. USA 74, 1865-1869. 3. Tomizawa, J., Sakakibara, Y. and Kakefuda, T. (1975) Proc. Natl. Acad. Sci. USA 72, 1050-1054. 4. Itoh, T. and Tomizawa, J. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 409-417.

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5. Itoh, T. and Tomizawa, J. (1980) Proc. Natl. Acad. Sci. USA 77, 2450- 2454. 6. Tomizawa, J., Itoh, T., Selzer, G. and Som, T. (1981) Proc. Natl. Acad. Sci. USA 78, 1421-1425. 7. Tomizawa, J. and Itoh, T. (1981) Proc. Natl. Acad. Sci. USA 78, 6096- 6100. 8. Gellert, M., O'Dea, M., Itoh, T. and Tomizawa, J. (1976) Proc. Natl. Acad. Sci. USA 73, 4474-4478. 9. Kindler, P., Keil, T. U. and Hofschneider, P. H. (1973) Molec. gen. Genet. 126, 53-59. 10. Apirion, D. and Watson, N. (1975) J. Bacteriol. 124, 317-324. 11. Dunn, J. J. and Studier, F. W. (1975) J. Molec. Biol. 99, 487-499. 12. Ohmori, H. and Tomizawa, J. (1979) Molec. gen. Genet. 176, 161-170. 13. Germond, J. E., Hirt, B., Oudet, P., Gross-Bellard, M. and Chamborn, P. (1975) Proc. Natl. Acad. Sci. USA 72, 1843-1847. 14. Darlix, J.-L. (1975) Eur. J. Biochem. 51, 369-376. 15. Arndt-Jovin, D., Jovin, T., Bfhr, W., Frischauf, A.-M. and Marquardt, M. (1975) Eur. J. Biochem. 54, 411-418. 16. Berkower, I., Leis, J. and Hurwitz, J. (1973) J. Biol. Chem. 248, 5914-5921. 17. Dunn, J. J. (1976) J. Biol. Chem. 251, 3807-3814. 18. Laemmli, U. (1970) Nature 227, 680-683. 19. BUlcher, T. (1947) Biochim. Biophys. Acta 1, 292-314. 20. McKnight, G. S. (1977) Anal. Biochem. 78, 86-92. 21. Vicuna, R., Hurwitz, J., Wallace, S. and Girard, M. (1977) J. Biol. Chem. 252, 2524-2531. 22. Levine, A. D. and Rupp, W. D. (1978) In "Microbiology 1978" (Schlessinger, D., ed.), pp. 163-166. Amer. Soc. Microbiology. 23. Blair, D. G., Sherratt, D. J., Clewell, D. B. and Helinski, D. R. (1972) Proc. Natl. Acad. Sci. USA 69, 2518-2522. 24. Sugino, Y., Tomizawa, J. and Kakefuda, T. (1975) Nature 253, 652-654. 25. Kahn, M., Figurski, D., Ito, L. and Helinski, D. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 99-103. 26. Conrad, S. E. and Campbell, J. L. (1979) Cell 18, 61-71. 27. Ely, S. and Staudenbauer, W. L. (1981) Molec. gen. Genet. 181, 29-35. 28. Arendes, J., Carl, P. L., Sugino, A. (1982) J. Biol. Chem. 257, 4719-4722. 29. Sumida-Yasumoto, C., Ikeda J.-E., Benz. E., Marians, K. J.,, Vicuna, R., Sugrue, S., Zipursky, S. L. and Hurwitz, J. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 311-329. 30. Miller, H . I., Riggs, A. D. and Gill, G. N. (1973) J. Biol. Chem. 248, 2621-2624. 31. Molineux, I. J., Friedman, S. and Gefter, M. L. (1974) J. Biol. Chem. 249, 6090-6098. 32. Hillenbrand, G. and Staudenbauer, W. L. (1982) Nucleic Acids Res. 10, 833-853. 33. Carl, P. L., Bloom, L. and Crouch, R. (1980) J. Bacteriol. 144, 28-35. 34. Bolivar, F., Betlach, M. C., Heynecker, H. L., Shine,XJ., Rodriguez, R. L. and Boyer, H. W. (1977) Proc. Natl. Acad. Sci. USA, 74, 5265-5269.

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