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 poly- merase 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 re- quired 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 differ- ent 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 mole- cules 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 plas- mid 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 pre- pared 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 forma- tion 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 pre- vious method invariably yielded products that contained RNase III activity de- tectable 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 differ- ent 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.