J. Biochem. 84, 385-393 (1978)
I. Partial Purification and Characterization of Nuclear Ribonuclease H 1 Fumio TASHIRO* and Yoshio UENO Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya, Shinjuku-ku, Tokyo 162 Received for publication, March 11, 1978
A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cell.
The existence of multiple forms of ribonuclease H has been reported in eukaryotes such as rat liver (7), a basidiomycete, Ustilago maydis (2), murine myeloma (3), calf thymus (4), yeast (5, 6), Ehrlich ascites cells (7), baby hamster kidney cell line (8),
1
This work was supported in part by grants from the Ministry of Education, Science and Culture (1976) and the Ministry of Public Health and Welfare (1976), Japan. 1 To whom request for reprint should be addressed. Enzymes: Ribonuclease H or RNA-DNA-hybnd nbonucleotidohydrolase [EC 3.1.4.34], DNA-dependent RNA polymerase or nucleosidetriphosphate: RNA nucleotidyltransferase [EC 2.7.7.6], Snake venom phosphodiesterase or oligonucleate 5'-nucleotidohydrolase [EC 3.1.4.1], Bovine pancreatic ribonuclease or ribonucleate 3'-pyrimidinooligonucleotidohydrolase [EC 3.1.4.22]. Vol. 84, No. 2, 1978
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Ribonuclease H from Rat Liver
and a protozoan, Tetrahymena pyriformis (9). However, it is still not clear where they are localized in the cell and how they are related with each other. As for rat liver ribonuclease H, two enzymes were detected in a 70,000 x g supernatant fraction (/) while only one species of the enzyme has been purified from rat liver cytosol and characterized (10). On the other hand, the nuclear enzyme of rat liver has not been studied in detail. In the present communication it is shown that one species of ribonuclease H occurs in rat liver nuclei. MATERIALS AND METHODS
Buffers—Buffer A: 50mM Tris-HCl (pH 7.9), 10% (v/v) glycerol, 5 mM MgCl,, 0.1 mM EDTA, and 0.5 mM dithiothreitol. Buffer B: 50 mM TrisHCl (pH7.9), 30% (v/v) glycerol, and 0.5 mM dithiothreitol.
386
prepared by incubating equimolar quantities of a [3H]poly(rU) together with poly(rA) or poly(dA) in 0.1 M NaCl and 0.01 M Tris-HCI (pH 7.5) at 30°C for 30 min (14). Assay of Ribonuclease H and Unit of Activity —The reaction mixture contained in a final volume of 300^1, 50 mM Tris-HCI (pH 7.9), 25 mM (NHJjSO*. 17 mM MgCl,, 0.5 mM dithiothreitol, [14C]RNA-DNA hybrid (2,000-2,500 cpm) and 50 fi\ of enzyme solution. After incubation at 37°C for 30 min, acid-insoluble material was precipitated by the addition of 200 ft\ of ice-cold 15% trichloroacetic acid and 100 ftl of yeast RNA (2 mg/ml) as co-precipitant. The mixture was centrifuged at 1,600 xg for 15 min. Radioactivity in the acid-soluble supernatant fraction was determined in a Packard Liquid Scintillation Spectrometer using Bray's scintillator (15). One unit of enzyme activity is defined as the amount of enzyme required to make 1 pmol of labelled ribonucleotide acid-soluble under the standard assay conditions described above. Isolation of Nuclei, Nucleoh, and Nucleoplasm —Nuclei were isolated from livers of three male Wistar rats (200-300 g) by the method of Higashinakagawa et al. (16). All procedures were carried out at below 4°C. The perfused livers were weighed, minced and homogenized with three up-and-down strokes in 10 vol. of 2.3 M sucroselOmM MgClt in a loosely fitting Potter-Elvehjem type homogenizer with a Teflon pestle. The homogenate was filtered through four layers of gauze and centrifuged at 40,000Xg for 1 h. The nuclear pellet was suspended in 0.34 M sucrose1 mM MgClj and centrifuged at low speed for 15 min. The nuclear pellet was used as the nuclear fraction for the preparation of enzyme. The nuclear pellet was suspended in 0.34 M sucrose-0.05 mM MgCl, and sonicated in 20 ml batches in a Tomy Ultra Sonic Generator Model UR-150P (Tominaga Works LTD) at 20 kc/s for 60s. The sonicate was rapidly checked for the extent of disruption of nuclei under a light microscope. When virtually all the nuclei were destroyed, the sonicate was layered on 20 ml of 0.88 M sucrose-0.05 mM MgCl, and centrifuged at 2,000 x g for 20 min in the cold room. The resulting pellet was used as nucleolar fraction and the supernatant as nucleoplasmic fraction.
J. Biochem.
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Chemicals—Sephadex G-50, G-150, G-200, and DEAE-Sephadex A-25 were obtained from Pharmacia and phosphocellulose from Whatman. [UC]UTP (59Ci/mol) and PHJthymidine (14 Ci/ mmol) were purchased from Radiochemical Centre (Amersham, England). ['HIPoly(rU) (7.76 Ci/ mol) was purchased from Schwarz. Unlabelled nucleotides were purchased from Boehringer Mannheim and Sigma. Polyribonucleotides and polydeoxyribonucleotides (molecular weight> 100,000) were purchased from Miles and Boehringer Mannheim. Calf thymus DNA was obtained from Sigma. 3H-Labelled T7 DNA was prepared according to the method of Davison and Freifelder (11). All other reagents were of analytical grade. Substrates—[14C]RNA-DNA hybrid was prepared with heat-denatured calf thymus DNA (100°C, 5 min) and rat liver RNA polymerase II isolated by the method of Roeder and Rutter (12). The reaction mixture contained in a final volume of 3 ml, 50 mM Tris-HCI (pH 7.9), 110 mM ammonium sulphate, 1.5 mM MnCI2, 4 pimol of ATP, 1 /Jmol each of GTP and CTP, 0.5 mM dithiothreitol, 400 ^g of heat-denatured calf thymus DNA, 2 fid of [UC]UTP (59Ci/mol) and 200 ^units of rat liver RNA polymerase II (13). After incubation at 37°C for 2 h, sodium dodecylsulphate was added to a final concentration of 0.5 % in order to denature the protein. The mixture was then passed through Sephadex G-50 (1.5 x52 cm) equilibrated with 0.1 M NaCl, in order to remove acid-soluble radioactive materials. The RNA-DNA hybrid was precipitated by addition of 3 vol. of ethanol in the presence of 500 ft% yeast RNA. The precipitate was collected by centrifugation, dissolved in 0.1 M NaCl and adjusted to a concentration of 18-23 pmol of nucleotides of synthetic RNA (2,000-2,500 cpm) per 50/il. Eighty-five to ninety % of the hybrid was resistant to ribonuclease A (5 pg/ml, 37°C, 10 min), but it was completely hydrolyzed by the same enzyme after heating at 100°C for 5 min followed by rapid cooling. The specific activity of hybrids was 2,240 cpm/^g RNA. ["QRNA[*H]DNA hybrid was prepared with heat-denatured [*H]T7 DNA (3,478 cpm/^g DNA) and rat liver RNA polymerase II under the conditions described above. The double-labelled hybrid was dissolved in 0.1 M NaCl and adjusted to 213 cpm of ["C]RNA and 1,874 cpm of fH]DNA per 50 pi. [»H]Poly(rU>poly(rA) and [*H]poly(rU>poly(dA) were
F. TASHIRO and Y. UENO
NUCLEAR RIBONUCLEASE H FROM RAT LIVER 10
387
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80.5
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50
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Fig. 1. DEAE-Sephadex chromatography of nbonuclease H and RNA polymerase. The crude extract was applied to DEAE-Sephadex column equilibrated with 0.05 M ( N H J ) ^ O 4 in buffer A (bed volume 16 ml, column size 1 6 x 8 cm, flow rate 15 ml/h). The column was washed with 0.05 M ( N H ^ J S O , in buffer A, then the adsorbed material is eluted with a 0.05-0.5 M (NH^.SO, linear gradient in buffer A (45 ml+45 ml). Fractions of 2.5 ml were collected and assayed for nbonuclease H and RNA polymerase activity. Assay procedure for RNA polymerase was reported previously (75). , Absorbance at 280 nm; O, nbonuclease H activity; • , RNA polymerase activity, , (NHJ.SO, concentration.
1000
RESULTS Solubilization and Purification of Ribonuclease H—Ribonuclease H was solubilized together with DNA-dependent RNA polymerase by sonication of the isolated nuclei, nucleoli or nucleoplasm in a medium of high ionic strength essentially according to the method of Roeder and Rutter (72, 13). The solubilized enzyme was first applied to the column of DEAE-Sephadex. DNA-dependent RNA polymerases I and II were eluted at around 0.15 M and 0.25 M of ammonium sulphate, respectively (Fig. 1). Ribonuclease H activity was detected in the flow-through region of the chromatography. The column fractions having ribonuclease H activity were combined. To the enzyme solution was added solid ammonium sulphate (0.42 g/ml) and the mixture was stirred in the cold room for 1 h. The precipitate was collected by centrifugation at 100,000 XQ for 1 h. The precipitate was dissolved in 4 ml of 0.5 M NH4CI in buffer A and dialyzed against the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column previously equilibrated with 0.5 M NH 4 Q buffer A and eluted with the same buffer Vol. 84, No. 2, 1978
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E 1500 u
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Fig. 2. Sephadex G-200 gel filtration of nbonuclease H. Solid (NH4),SO4 (0.42 g/ml) was added to the combined pools of the DEAE-Sephadex chromatography. The precipitate formed was collected by centnfugation at 100,000x(/ for 1 h, dissolved in 4ml of 0.5 M NH4C1 in buffer A, and dialyzed against 300 ml of the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column equilibrated with the same buffer (bed volume 182 ml, column size 2x58 cm, flow rate 9 ml/h, fraction volume 2.8 ml). Ribonuclease activity was assayed with heat-denatured [14C]RNADNA hybnd as substrate by determining acid-soluble radioactivity. , Absorbance at 280 nm; O, ribonuclease H activity; • , ribonuclease activity.
F. TASfflRO and Y. UENO
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60 30 10 50 Fraction number
70
80
Fig. 3. Sephadex G-150 gelfiltrationof nbonuclease H. The combined pools of Sephadex G-200 chromatography were dialyzed overnight against buffer A containing 0.1 M NH4C1 and 30% (w/v) polyethyleneglycol. The dialysate (2.5 ml) was applied to a Sephadex G-150 column previously equilibrated with 0.1 M NH4CI in buffer A and eluted with the same buffer (bed volume 182 ml, column size 2x58 cm,flowrate 6 ml/h, fraction volume 2 ml). , Absorbance at 280 nm; o , ribonuclease H activity. (Fig. 2). The fractions having ribonuclease H activity were collected and dialyzed against 0.1 M NH4CI and 30% (w/v) polyethyleneglycol in buffer A overnight. The dialysate was applied to a Sephadex G-150 column previously equilibrated with 0.1 M NH4C1 in buffer A and eluted with the same buffer (Fig. 3). This chromatographic step resulted in the further removal of non-active protein from the enzyme preparation. The ribonuclease H fractions were collected and dialyzed against 1,000 ml of 0.05 M NH4C1 in buffer B. The dialysate was applied to a phosphocellulose column previously equilibrated with 0.05 M NH 4 Q in buffer B. The column was washed exhaustively with the same buffer, then with a linear gradient from 0.05 M to 1.0 M NH4C1 in buffer B (Fig. 4). The nucleolytic activity was separated in two peaks, one corresponding to a ribonuclease H that degrades ["C]RNA-DNA hybrid and the other to an enzyme that degrades only heat-denatured P*C]RNA-DNA hybrid. The fractions having ribonuclease H activity were stored at —20°C in 50% (v/v) glycerol without loss of activity at least several months. A summary of the purification procedure is presented in Table I. The specific activity of the final purified preparation was 9 times that of the crude extract. Substrate Specificity—To determine the substrate specificity of the enzyme, RNA-DNA hybrid
was replaced with other nucleic acid substrates. The results are shown in Table n . Ribonuclease H degraded the RNA moiety of RNA-DNA hybrid, while single or double-stranded T7 DNA, 1000
10
20 30 «> Fraction number
Fig. 4. Phosphocellulose chromatography of nbonuclease H. The nbonuclease H activity obtained by Sephadex G-150 chromatography was applied to a column of phosphocellulose equilibrated with 0.05 M NH4C1 in buffer B (bed volume 5 ml, column size 0.8X10 cm. flow rate lOml/h). The column was washed with 0.05 M NH4C1 in buffer B, then with a 0.05-1.0 M NH4C1 linear gradient in buffer B (30ml + 30 ml). Fractions of 2 ml were collected and assayed for ribonuclease H activity and ribonuclease activity under the standard assay conditions. , Absorbance at 280 nm; C, ribonuclease H activity; • , ribonuclease activity; , NH4C1 concentration.
/. Biochem.
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10
NUCLEAR RIBONUCLEASE H FROM RAT LIVER
Steps Crude extract DEAE-Sephadex Sephadex G-200 Sephadex G-150 Phosphocellulose
Total protein (mg)
Specific activity (units//* g)
Total activity (units)
65.9 16.9 12.3
1.38 4.26
91,228 72,000
5.49 7.39 12.22
67,459 72,786 636
9.9
0.052
TABLE II. Substrate specificity of ribonuclease H of rat liver nuclei. The following amounts of substrates were assayed under standard assay conditions: 1,335 cpm of native or 1,691 cpm of denatured P*C]RNA-DNA hybrid (1,156 cpm//*g RNA); 669 cpm of native or 722 cpm of denatured PH]T7 DNA (3,478 cpm//*g DNA); 276 cpm of PH]pory(rU) (7.76 Ci/mol); 975 cpm of [»H]poly(rU>poly(dA); 352 cpm of PH]poly(rU>poly(rA). For denaturation of RNA-DNA hybrid and T7 DNA, the samples were heated at 100°C for 5 min and quickly chilled in ice. Amount of enzyme used was 8.3 units. Substrate
Acid-soluble radioactivity (%)
P*C]RNA-DNA hybrid P*C]RNA-DNA hybrid, denatured at 100°C for 5 mm 'H-Labelled T7 DNA l H-Labelled T7 DNA, denatured at 100°C for 5 min PH]Poly(rU) PH]Poly(rU>poly(rA)
68.8
PH]Poly(rU)-poly(dA)
17.7
0.4 2.0 0.0 0.7 0.0
poly(rU) and poly(rU)-poly(rA) duplex were hardly affected. Poly(rU>poly(dA) was degraded to some degree. To examine whether the DNA in the hybrid is sensitive to the enzyme, ["QRNA-PHJDNA hybrid was subjected to degradation by the enzyme. The " C radioactivity in RNA was progressively rendered acid-soluble as the incubation proceeded, whereas the SH radioactivity in DNA remained Vol. 84, No. 2, 1978
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20 Time (mm )
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TABLE I. Summary of the purification procedure for nuclear ribonuclease H. The reaction mixture for the ribonuclease H was described in detail in " MATERIALS AND METHODS." The amount of protein was determined by the method of Lowry et al. (35).
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Fig. 5. Sensitivity of double-labelled hybrid to ribonuclease H. ["C]RNA-r*H]DNA hybrid was synthesized as described in " MATERIALS AND METHODS." Samples containing 213 cpm of ["QRNA and 1,874 cpm of PH]DNA were incubated with the enzyme (0.6 units) for various lengths of time. O, "C Radioactivity; • , SH radioactivity.
acid-precipitable, as shown in Fig. 5. Therefore, it is apparent that the enzyme degrades specifically the RNA region of RNA-DNA hybrid. Optimal Conditions for Ribonuclease H Activity
—The optimal conditions for the ribonuclease H reaction were investigated by varying the standard assay procedure described in " MATERIALS AND METHODS." [14C]RNA-DNA hybrid was incubated together with the enzyme under conditions specified in the legend of Fig. 6. The optimal conditions for the enzyme reaction with respect to pH, ionic strength and divalent cations were demonstrated. The enzyme was most active at pH 8.5-9.0. Optimal concentration was 25 mM for (NHJ.SO,, 17 mM for Mg1+, and 0.6 mM for Mn1+. Properties of the Purified Enzyme—The molecular weight of the enzyme was calculated to be about 36,000 by Sephadex G-200 gel filtration (Fig. 7). We investigated effects of various nucleic acid on the ribonuclease H reaction (Table III). Single and double-stranded DNA showed very strong inhibition, while polyribonucleotides exhibited only slight inhibition. Mode of Action of Ribonuclease H—To elucidate the mode of action, ["QRNA-DNA hybrid was digested to various extents and the reaction products were chromatographed under conditions
F. TASHIRO and Y. UENO
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9 pH
10 0 25 JO 100 200 Concnof (NH^)2SOA (mM)
15
t/i
a
"5200
200 10
Molecular weight 100
0 10 20 0 1 2 3 A Concn of MgCl2 (mM) Coocn of MnCl2 (mM)
5
Fig. 6. Optimal concentrations for ribonuclease H activity. The experiments were earned out under the standard assay conditions with various pHs and concentrations of (NH4),SO4, MgCl,, and MnCl2. (A) Optimal pH of the enzyme. Tris-HCl buffer (50 mM) was used from pH 7.5 to 9.0, glycine-NaOH buffer (50 mM) from pH 9.0 to 10.0; (B) effect of ionic strength on the enzyme; ( Q dependency of the enzyme on MgCl t ; (D) dependency of the enzyme on MnCl,. Amount of enzyme used in experiments (A), (B), (C), and (D) was 3.5, 4 8, 1.6, and 1.6 units, respectively.
under which only small oligonucleotides would migrate (9, 17). At various stages of digestion by the enzyme, the bulk of the products remained as large molecules (Fig. 8, A-C). Even when 63% of the RNA moiety had been rendered acid-soluble, the amount of UMP was found to be very small. The data obviously indicate that the enzyme is an endonuclease. The resulting oligonucleotides were completely converted into 5'-UMP by snake venom phosphodiesterase (Fig. 8, D). The results strongly suggest that the oligonucleotides produced by the ribonuclease H of rat liver nuclei terminate in a 3'-hydroxyl group.
Fig. 7. Molecular weight of ribonuclease H determined by gel filtration. A published procedure was essentially followed (33). The column was 2x58 cm; the gel was Sephadex G-200; the elution buffer 0.5 M NH4C1 in buffer A. All operations were carried out in the manner described under Fig. 2. Myoglobin, chymotrypsinogen, ovalbumin, bovine serum albumin and ^-globulin (Schwarz/Mann) were detected by absorbance at 280 nm. Rat liver nuclear nbonuclease H was detected by activity measurement. Vo represents the void volume (estimated with blue dextran). Ke represents the elution volume.
TABLE HI. Effects of nucleic acids on ribonuclease H of rat liver nuclei. To the enzyme preincubated with each nucleic acid for 3 min at 37°C was added P4C]RNA-DNA hybrid and the mixture was incubated for 10 min at 37°C. The reaction was stopped by addition of 200 fi\ of 15% TCA and 100 y\ of yeast RNA (2 mg/ml) and the acid-soluble radioactivity was assayed as described in " MATERIALS AND METHODS." Amount of enzyme used was 6.7 units. Activity ([% of control) 12.5
/ig/assay 25 50 100
87
88
86
86
85
Poly(rA)
86
104
105
93
94
Poly(rC)
93
99
85
88
84
Calf thymus DNA (native)
7
3
0
0
0
Calf thymus DNA (denatured)
0
0
0
0
0
Nucleic acid
Poly(rU)
200
J. Biochem.
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NUCLEAR RIBONUCLEASE H FROM RAT LIVER
3000
391
B ( 477. )
A ( 0 V. )
1000
I 1000
1000 C ( 63V. )
D ( 637. )
pU
500
0
10 20 30 0 10 Distance from origin ( cm )
Fig. 8. Chromatography of digestion products of rat liver nuclear ribonuclease H. ["QRNA-DNA hybrid (3,897 cpm) with labelled UMP residues was incubated at 37°C for various lengths of time with the enzyme (43.3 units). The extent of degradation, determined by the standard assay, is indicated in parentheses. The reaction mixture was subjected to descending paper chromatography in the manner described previously (9). One-centimeter strips were cut from the chromatogram and assayed for radioactivity. O represents the origin, pU the position of 5'-UMP, and U the position of uridine. A) Unreacted hybrid, B) digestion for 7 min, Q digestion for 15 min, D) digestion for 15 min followed by treatment of half of sample with snake venom phosphodiesterase for 30 min at 37°C.
20 30 Fraction number
Fig. 9. DEAE-Sephadex chromatography of nucleoplasmic and nucleolar ribonuclease H and RNA polymerase. The crude extract was applied to the DEAESephadex A-25 column in the manner described under Fig. 1. , (NH4),SO4 concentration; O, ribonuclease H activity; • , RNA polymerase activity. A) The crude extract from nucleoplasmic fraction, B) the crude extract from nucleolar fraction.
The RNA polymerase activity eluting at 0.15 M (NH4),SO4 on DEAE-Sephadex chromatography was completely insensitive to a-amanitin (1 /ig/ml), while the RNA polymerase activity eluting at 0.25 M (NH4),SO4 was completely sensitive to low concentration of a-amanitin (1 /^g/ml). We concluded that the former enzyme was RNA polymerase I and the latter RNA polymerase II (18). The Intranuclear Localization of the Enzyme—To preparation of nucleoplasm hardly exhibited any investigate the localization of the enzyme in the RNA polymerase I activity in all experiments. nucleus, we extracted the enzymes from the nucle- Although the preparation of nucleoli contained oplasm and nucleoli and compared their properties. some RNA polymerase II activity (Fig. 9), the It is well known that DNA-dependent RNA large amount of ribonuclease H activity contained polymerases I and II exist in nucleoli and nucle- in the preparation of nucleoli could not be acoplasm, respectively. Therefore, we checked the counted for by contamination by the nucleoplasm. purity of our preparations of nucleoli and nucleThe two ribonuclease H preparations from the oplasm on the basis of their contents of these two nucleoli and nucleoplasm could not be distinguished RNA polymerase activities. from each other on the basis of such properties as Vol. 84, No. 2, 1978
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DISCUSSION We have partially purified and characterized the nuclear ribonuclease H of rat liver. As summarized in Table I, the purification with phosphocellulose chromatography resulted in an inexplicable loss of the enzyme activity. The introduction of more effective purification procedures such as DNA-cellulose chromatography is expected to increase the yield of the enzyme activity. The properties of rat liver nuclear ribonuclease H were distinct from those of rat liver cytosol enzyme reported by Roewekamp and Sekeris (10) in many respects, such as molecular weight, optimal pH and requirements for Mgs+ and Mn'+ ions. As reported in the accompanying paper, we prepared the rat liver cytosol enzyme by the method of Roewekamp and Sekeris (10) and confirmed these differences between the nuclear and cytosol enzymes. Multiple forms of ribonuclease H have been reported in various eukaryotic organisms (1-9). These reports and our present results imply that multiple forms of ribonuclease H are present in different regions of the cell in higher organisms. As for its substrate specificity, the nuclear enzyme rapidly hydrolyzed up to 65 % of the RNA moiety of RNA-DNA hybrid but not the RNA dissociated from the hybrid by heat denaturation. Degradation of single or double-stranded DNA, poly(rU) and poly(rU)-poly(rA) was negligibly low. Poly(rU)-poly(dA) showed a poor substrate activity. In the presence of Mg1+ ions, the nuclear enzyme was very similar in its action on poly(rU)poly(dA) to the ribonuclease H isolated from rat liver cytosol (10), Ustilago maydis (3), human leukemic blood cells (19), and calf thymus (4), but different from that of KB-cells (20). Analysis of the reaction products demonstrated that the enzyme cleaves the RNA chain endonucleolytically producing oligonucleotides with 3'-hydroxyl termini. This mode of action is similar to that
of ribonuclease H of other cellular origins (2, 10, 14, 20-22). In the presence of polyribonucelotides the enzyme activity was slightly inhibited, but the inhibition was complete in the presence of single or double-stranded DNA. The same results were also obtained in the case of Raucscher leukemia virus ribonuclease H (23). These results strongly suggest that ribonuclease H first binds to the DNA region of RNA-DNA hybrid, recognizes the RNA of the hybrid and finally degrades the RNA region. When RNA-DNA hybrid used as substrate is contaminated with free RNA or single-stranded DNA and the enzyme preparation contains other nucleases, the exact determination of ribonuclease H activity is very difficult. For this purpose it is preferable to use the RNA-DNA hybrid treated with SI nuclease (34). The existence of multiple forms of ribonuclease H in rat liver and other organisms raises the question of the biological function of the enzymes as well as of their substrate, the RNA-DNA hybrid. The demonstration of ribonuclease H in the nucleus strongly corroborates the possible role of this enzyme in the metabolic process of nucleic acid. Several authors have indicated that ribonuclease H might be involved in the removal of the initiator RNA chain priming DNA synthesis (24-28), while others have suggested a possible role in the process of transcription (7, 29-31). Additionally, Wyers et at. have postulated a possible role in the chromatin structure (6). The existence of only one species of nuclear ribonuclease H suggests that the same kind of ribonuclease H functions in both ribosomal RNA and heterogeneous nuclear RNA synthesis although different forms of DNA-dependent RNA polymerase are involved in the respective processes. Blisen et al. have reported that ribonuclease H lib increased in parallel with RNA synthesis and ribonuclease H I increased together with DNA synthesis during the response of bovine lymphocytes to concanavalin A (32). It is still premature to define the real role of ribonuclease H. However these observations point to the interesting possibility that ribonuclease H plays a crucial role in the metabolism of nucleic acid.
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optimal concentrations of divalent cations, sensitivity to rifampicin derivatives, behavior on DEAESephadex and phosphocellulose column chromatography. From these results, we infer that the ribonuclease H of rat liver nuclei is only one species and exists in both nucleoplasm and nucleoli.
NUCLEAR RIBONUCLEASE H FROM RAT LIVER
REFERENCES 1. Sekeris, C.E. & Roewekamp, W. (1972) FEBS Lett. 23, 34-36 2. Banks, G.R. (1974) Eur. J. Biochem. 47, 499-507 3. O'Cuinn, G., Persico, F.J., & Gottlieb, A.A. (1973) Biochim. Biophys. Ada 324, 78-85 4. BUsen, W. & Hausen, P. (1975) Eur. J. Biochem. 52, 179-190 5. Wyers, F., Sentenac, A., & Fromageot, P. (1976) Eur. J. Biochem. 69, 377-383 6. Wyers, F., Huet, J., Sentenac, A., & Fromageot, P. (1976) Eur. J. Biochem. 69, 385-395 7. Naton, S., Takeuchi, K., Takahashi, K., & Mizuno, D. (1973) /. Biochem. 73, 879-888 8 Cooper, R.J., Duff, P.M., Oliver, A., Craig, R.K., & Keir, H.M. (1974) FEBS Lett. 45, 38-^3 9. Tashiro, F., Mita, T., & Higashinakagawa, T (1976) Eur. J. Biochem. 65, 123-130 10. Roewekamp, W. & Sekeris, C.E. (1974) Eur. J. Biochem. 43, 405-413 11. Davison, P.F. & Freifelder, D. (1962) /. Mol. Bwl. 5, 635-642 12. Roeder, R.G. & Rutter, W.J. (1970) Proc. Natl. Acad. Sci. U.S. 65, 675-682 13. Higashinakagawa, T., Tashiro, F., & Mita, T. (1975) J. Biochem. TJ, 783-793 14. Harberkern, R.C. & Cantoni, G.L. (1973) Biochemistry 12, 2389-2395 15. Bray, G.A. (1960) Anal. Biochem. 1, 279-285 16. Higashinakagawa, T., Muramatsu, M., & Sugano, H. (1972) Exptl. Cell Res. 71, 65-74
Vol. 84, No. 2, 1978
17. Lapidot, Y. & Khorana, H.G. (1963) J. Am. Chem. Soc. 85, 3857-3862 18. Weimann, R. & Roeder, R.G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1790-1794 19. Sargadharan, M.G., Leis, J.P., & Gallo, R.C. (1975) /. Biol. Chem. 250, 365-373 20. Keller, W. & Crouch, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3360-3364 21. Leis, J.P., Bcrkower, I., & Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U.S. 70, 466-470 22. Miller, H.I., Riggs, A.D., & Gill, G.N. (1973) / . Biol. Chem 248, 2621-2624 23. Modak, M.J. & Marcus, S.L. (1977) / . Virol. 22, 243-246 24. Wickner, W., Brutlag, D., Schekman, R., & Komberg, A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 965-969 25. Lark, K.G. (1972) J. Mol. Biol. 65, 47-60 26. Sugino, A., Hirose, S., & Okazaki, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1863-1867 27. Keller, W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1560-1564 28. Spadari, S. & Weissbach, A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 503-507 29. Sekeris, C.E., Schmid, W., & Roewekamp, W. (1972) FEBS Lett. 24, 27-31 30. Huet, J., Wyers, F., Buhler, J.M., Sentenac, A., & Fromageot, P. (1976) Nature 261, 431-433 31. Doenecke, D., Marmaras, V.J., & Sekeris, C.E. (1972) FEBS Lett. 11, 261-264 32. BUsen, W., Peters, J.H., & Hausen, P. (1977) Eur. J. Biochem. 74, 203-208 33. Andrews, P. (1964) Biochem. J. 91, 222-233 34. Sutton, W.D. (1971) Biochim. Biophys. Ada 240, 522-531 35. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) /. Biol. Chem. 193, 265-275
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We wish to thank Dr. T. Mita of National Cancer Center Research Institute and Dr. T. Higashinakagawa of Mitsubishi-Kasei Institute of Life Sciences for helpful discussions and Drs. T. Ando and E. Hayase of The Institute of Physical and Chemical Research for preparation of 'H-Iabelled T7 DNA.
393
I. Partial Purification and Characterization of Nuclear Ribonuclease H 1 Fumio TASHIRO* and Yoshio UENO Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya, Shinjuku-ku, Tokyo 162 Received for publication, March 11, 1978
A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cell.
The existence of multiple forms of ribonuclease H has been reported in eukaryotes such as rat liver (7), a basidiomycete, Ustilago maydis (2), murine myeloma (3), calf thymus (4), yeast (5, 6), Ehrlich ascites cells (7), baby hamster kidney cell line (8),
1
This work was supported in part by grants from the Ministry of Education, Science and Culture (1976) and the Ministry of Public Health and Welfare (1976), Japan. 1 To whom request for reprint should be addressed. Enzymes: Ribonuclease H or RNA-DNA-hybnd nbonucleotidohydrolase [EC 3.1.4.34], DNA-dependent RNA polymerase or nucleosidetriphosphate: RNA nucleotidyltransferase [EC 2.7.7.6], Snake venom phosphodiesterase or oligonucleate 5'-nucleotidohydrolase [EC 3.1.4.1], Bovine pancreatic ribonuclease or ribonucleate 3'-pyrimidinooligonucleotidohydrolase [EC 3.1.4.22]. Vol. 84, No. 2, 1978
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Ribonuclease H from Rat Liver
and a protozoan, Tetrahymena pyriformis (9). However, it is still not clear where they are localized in the cell and how they are related with each other. As for rat liver ribonuclease H, two enzymes were detected in a 70,000 x g supernatant fraction (/) while only one species of the enzyme has been purified from rat liver cytosol and characterized (10). On the other hand, the nuclear enzyme of rat liver has not been studied in detail. In the present communication it is shown that one species of ribonuclease H occurs in rat liver nuclei. MATERIALS AND METHODS
Buffers—Buffer A: 50mM Tris-HCl (pH 7.9), 10% (v/v) glycerol, 5 mM MgCl,, 0.1 mM EDTA, and 0.5 mM dithiothreitol. Buffer B: 50 mM TrisHCl (pH7.9), 30% (v/v) glycerol, and 0.5 mM dithiothreitol.
386
prepared by incubating equimolar quantities of a [3H]poly(rU) together with poly(rA) or poly(dA) in 0.1 M NaCl and 0.01 M Tris-HCI (pH 7.5) at 30°C for 30 min (14). Assay of Ribonuclease H and Unit of Activity —The reaction mixture contained in a final volume of 300^1, 50 mM Tris-HCI (pH 7.9), 25 mM (NHJjSO*. 17 mM MgCl,, 0.5 mM dithiothreitol, [14C]RNA-DNA hybrid (2,000-2,500 cpm) and 50 fi\ of enzyme solution. After incubation at 37°C for 30 min, acid-insoluble material was precipitated by the addition of 200 ft\ of ice-cold 15% trichloroacetic acid and 100 ftl of yeast RNA (2 mg/ml) as co-precipitant. The mixture was centrifuged at 1,600 xg for 15 min. Radioactivity in the acid-soluble supernatant fraction was determined in a Packard Liquid Scintillation Spectrometer using Bray's scintillator (15). One unit of enzyme activity is defined as the amount of enzyme required to make 1 pmol of labelled ribonucleotide acid-soluble under the standard assay conditions described above. Isolation of Nuclei, Nucleoh, and Nucleoplasm —Nuclei were isolated from livers of three male Wistar rats (200-300 g) by the method of Higashinakagawa et al. (16). All procedures were carried out at below 4°C. The perfused livers were weighed, minced and homogenized with three up-and-down strokes in 10 vol. of 2.3 M sucroselOmM MgClt in a loosely fitting Potter-Elvehjem type homogenizer with a Teflon pestle. The homogenate was filtered through four layers of gauze and centrifuged at 40,000Xg for 1 h. The nuclear pellet was suspended in 0.34 M sucrose1 mM MgClj and centrifuged at low speed for 15 min. The nuclear pellet was used as the nuclear fraction for the preparation of enzyme. The nuclear pellet was suspended in 0.34 M sucrose-0.05 mM MgCl, and sonicated in 20 ml batches in a Tomy Ultra Sonic Generator Model UR-150P (Tominaga Works LTD) at 20 kc/s for 60s. The sonicate was rapidly checked for the extent of disruption of nuclei under a light microscope. When virtually all the nuclei were destroyed, the sonicate was layered on 20 ml of 0.88 M sucrose-0.05 mM MgCl, and centrifuged at 2,000 x g for 20 min in the cold room. The resulting pellet was used as nucleolar fraction and the supernatant as nucleoplasmic fraction.
J. Biochem.
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Chemicals—Sephadex G-50, G-150, G-200, and DEAE-Sephadex A-25 were obtained from Pharmacia and phosphocellulose from Whatman. [UC]UTP (59Ci/mol) and PHJthymidine (14 Ci/ mmol) were purchased from Radiochemical Centre (Amersham, England). ['HIPoly(rU) (7.76 Ci/ mol) was purchased from Schwarz. Unlabelled nucleotides were purchased from Boehringer Mannheim and Sigma. Polyribonucleotides and polydeoxyribonucleotides (molecular weight> 100,000) were purchased from Miles and Boehringer Mannheim. Calf thymus DNA was obtained from Sigma. 3H-Labelled T7 DNA was prepared according to the method of Davison and Freifelder (11). All other reagents were of analytical grade. Substrates—[14C]RNA-DNA hybrid was prepared with heat-denatured calf thymus DNA (100°C, 5 min) and rat liver RNA polymerase II isolated by the method of Roeder and Rutter (12). The reaction mixture contained in a final volume of 3 ml, 50 mM Tris-HCI (pH 7.9), 110 mM ammonium sulphate, 1.5 mM MnCI2, 4 pimol of ATP, 1 /Jmol each of GTP and CTP, 0.5 mM dithiothreitol, 400 ^g of heat-denatured calf thymus DNA, 2 fid of [UC]UTP (59Ci/mol) and 200 ^units of rat liver RNA polymerase II (13). After incubation at 37°C for 2 h, sodium dodecylsulphate was added to a final concentration of 0.5 % in order to denature the protein. The mixture was then passed through Sephadex G-50 (1.5 x52 cm) equilibrated with 0.1 M NaCl, in order to remove acid-soluble radioactive materials. The RNA-DNA hybrid was precipitated by addition of 3 vol. of ethanol in the presence of 500 ft% yeast RNA. The precipitate was collected by centrifugation, dissolved in 0.1 M NaCl and adjusted to a concentration of 18-23 pmol of nucleotides of synthetic RNA (2,000-2,500 cpm) per 50/il. Eighty-five to ninety % of the hybrid was resistant to ribonuclease A (5 pg/ml, 37°C, 10 min), but it was completely hydrolyzed by the same enzyme after heating at 100°C for 5 min followed by rapid cooling. The specific activity of hybrids was 2,240 cpm/^g RNA. ["QRNA[*H]DNA hybrid was prepared with heat-denatured [*H]T7 DNA (3,478 cpm/^g DNA) and rat liver RNA polymerase II under the conditions described above. The double-labelled hybrid was dissolved in 0.1 M NaCl and adjusted to 213 cpm of ["C]RNA and 1,874 cpm of fH]DNA per 50 pi. [»H]Poly(rU>poly(rA) and [*H]poly(rU>poly(dA) were
F. TASHIRO and Y. UENO
NUCLEAR RIBONUCLEASE H FROM RAT LIVER 10
387
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Fig. 1. DEAE-Sephadex chromatography of nbonuclease H and RNA polymerase. The crude extract was applied to DEAE-Sephadex column equilibrated with 0.05 M ( N H J ) ^ O 4 in buffer A (bed volume 16 ml, column size 1 6 x 8 cm, flow rate 15 ml/h). The column was washed with 0.05 M ( N H ^ J S O , in buffer A, then the adsorbed material is eluted with a 0.05-0.5 M (NH^.SO, linear gradient in buffer A (45 ml+45 ml). Fractions of 2.5 ml were collected and assayed for nbonuclease H and RNA polymerase activity. Assay procedure for RNA polymerase was reported previously (75). , Absorbance at 280 nm; O, nbonuclease H activity; • , RNA polymerase activity, , (NHJ.SO, concentration.
1000
RESULTS Solubilization and Purification of Ribonuclease H—Ribonuclease H was solubilized together with DNA-dependent RNA polymerase by sonication of the isolated nuclei, nucleoli or nucleoplasm in a medium of high ionic strength essentially according to the method of Roeder and Rutter (72, 13). The solubilized enzyme was first applied to the column of DEAE-Sephadex. DNA-dependent RNA polymerases I and II were eluted at around 0.15 M and 0.25 M of ammonium sulphate, respectively (Fig. 1). Ribonuclease H activity was detected in the flow-through region of the chromatography. The column fractions having ribonuclease H activity were combined. To the enzyme solution was added solid ammonium sulphate (0.42 g/ml) and the mixture was stirred in the cold room for 1 h. The precipitate was collected by centrifugation at 100,000 XQ for 1 h. The precipitate was dissolved in 4 ml of 0.5 M NH4CI in buffer A and dialyzed against the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column previously equilibrated with 0.5 M NH 4 Q buffer A and eluted with the same buffer Vol. 84, No. 2, 1978
(0
£ 500,
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70
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E 1500 u
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Fig. 2. Sephadex G-200 gel filtration of nbonuclease H. Solid (NH4),SO4 (0.42 g/ml) was added to the combined pools of the DEAE-Sephadex chromatography. The precipitate formed was collected by centnfugation at 100,000x(/ for 1 h, dissolved in 4ml of 0.5 M NH4C1 in buffer A, and dialyzed against 300 ml of the same buffer for 4 h. The dialysate was applied to a Sephadex G-200 column equilibrated with the same buffer (bed volume 182 ml, column size 2x58 cm, flow rate 9 ml/h, fraction volume 2.8 ml). Ribonuclease activity was assayed with heat-denatured [14C]RNADNA hybnd as substrate by determining acid-soluble radioactivity. , Absorbance at 280 nm; O, ribonuclease H activity; • , ribonuclease activity.
F. TASfflRO and Y. UENO
388
60 30 10 50 Fraction number
70
80
Fig. 3. Sephadex G-150 gelfiltrationof nbonuclease H. The combined pools of Sephadex G-200 chromatography were dialyzed overnight against buffer A containing 0.1 M NH4C1 and 30% (w/v) polyethyleneglycol. The dialysate (2.5 ml) was applied to a Sephadex G-150 column previously equilibrated with 0.1 M NH4CI in buffer A and eluted with the same buffer (bed volume 182 ml, column size 2x58 cm,flowrate 6 ml/h, fraction volume 2 ml). , Absorbance at 280 nm; o , ribonuclease H activity. (Fig. 2). The fractions having ribonuclease H activity were collected and dialyzed against 0.1 M NH4CI and 30% (w/v) polyethyleneglycol in buffer A overnight. The dialysate was applied to a Sephadex G-150 column previously equilibrated with 0.1 M NH4C1 in buffer A and eluted with the same buffer (Fig. 3). This chromatographic step resulted in the further removal of non-active protein from the enzyme preparation. The ribonuclease H fractions were collected and dialyzed against 1,000 ml of 0.05 M NH4C1 in buffer B. The dialysate was applied to a phosphocellulose column previously equilibrated with 0.05 M NH 4 Q in buffer B. The column was washed exhaustively with the same buffer, then with a linear gradient from 0.05 M to 1.0 M NH4C1 in buffer B (Fig. 4). The nucleolytic activity was separated in two peaks, one corresponding to a ribonuclease H that degrades ["C]RNA-DNA hybrid and the other to an enzyme that degrades only heat-denatured P*C]RNA-DNA hybrid. The fractions having ribonuclease H activity were stored at —20°C in 50% (v/v) glycerol without loss of activity at least several months. A summary of the purification procedure is presented in Table I. The specific activity of the final purified preparation was 9 times that of the crude extract. Substrate Specificity—To determine the substrate specificity of the enzyme, RNA-DNA hybrid
was replaced with other nucleic acid substrates. The results are shown in Table n . Ribonuclease H degraded the RNA moiety of RNA-DNA hybrid, while single or double-stranded T7 DNA, 1000
10
20 30 «> Fraction number
Fig. 4. Phosphocellulose chromatography of nbonuclease H. The nbonuclease H activity obtained by Sephadex G-150 chromatography was applied to a column of phosphocellulose equilibrated with 0.05 M NH4C1 in buffer B (bed volume 5 ml, column size 0.8X10 cm. flow rate lOml/h). The column was washed with 0.05 M NH4C1 in buffer B, then with a 0.05-1.0 M NH4C1 linear gradient in buffer B (30ml + 30 ml). Fractions of 2 ml were collected and assayed for ribonuclease H activity and ribonuclease activity under the standard assay conditions. , Absorbance at 280 nm; C, ribonuclease H activity; • , ribonuclease activity; , NH4C1 concentration.
/. Biochem.
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10
NUCLEAR RIBONUCLEASE H FROM RAT LIVER
Steps Crude extract DEAE-Sephadex Sephadex G-200 Sephadex G-150 Phosphocellulose
Total protein (mg)
Specific activity (units//* g)
Total activity (units)
65.9 16.9 12.3
1.38 4.26
91,228 72,000
5.49 7.39 12.22
67,459 72,786 636
9.9
0.052
TABLE II. Substrate specificity of ribonuclease H of rat liver nuclei. The following amounts of substrates were assayed under standard assay conditions: 1,335 cpm of native or 1,691 cpm of denatured P*C]RNA-DNA hybrid (1,156 cpm//*g RNA); 669 cpm of native or 722 cpm of denatured PH]T7 DNA (3,478 cpm//*g DNA); 276 cpm of PH]pory(rU) (7.76 Ci/mol); 975 cpm of [»H]poly(rU>poly(dA); 352 cpm of PH]poly(rU>poly(rA). For denaturation of RNA-DNA hybrid and T7 DNA, the samples were heated at 100°C for 5 min and quickly chilled in ice. Amount of enzyme used was 8.3 units. Substrate
Acid-soluble radioactivity (%)
P*C]RNA-DNA hybrid P*C]RNA-DNA hybrid, denatured at 100°C for 5 mm 'H-Labelled T7 DNA l H-Labelled T7 DNA, denatured at 100°C for 5 min PH]Poly(rU) PH]Poly(rU>poly(rA)
68.8
PH]Poly(rU)-poly(dA)
17.7
0.4 2.0 0.0 0.7 0.0
poly(rU) and poly(rU)-poly(rA) duplex were hardly affected. Poly(rU>poly(dA) was degraded to some degree. To examine whether the DNA in the hybrid is sensitive to the enzyme, ["QRNA-PHJDNA hybrid was subjected to degradation by the enzyme. The " C radioactivity in RNA was progressively rendered acid-soluble as the incubation proceeded, whereas the SH radioactivity in DNA remained Vol. 84, No. 2, 1978
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10
20 Time (mm )
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TABLE I. Summary of the purification procedure for nuclear ribonuclease H. The reaction mixture for the ribonuclease H was described in detail in " MATERIALS AND METHODS." The amount of protein was determined by the method of Lowry et al. (35).
389
Fig. 5. Sensitivity of double-labelled hybrid to ribonuclease H. ["C]RNA-r*H]DNA hybrid was synthesized as described in " MATERIALS AND METHODS." Samples containing 213 cpm of ["QRNA and 1,874 cpm of PH]DNA were incubated with the enzyme (0.6 units) for various lengths of time. O, "C Radioactivity; • , SH radioactivity.
acid-precipitable, as shown in Fig. 5. Therefore, it is apparent that the enzyme degrades specifically the RNA region of RNA-DNA hybrid. Optimal Conditions for Ribonuclease H Activity
—The optimal conditions for the ribonuclease H reaction were investigated by varying the standard assay procedure described in " MATERIALS AND METHODS." [14C]RNA-DNA hybrid was incubated together with the enzyme under conditions specified in the legend of Fig. 6. The optimal conditions for the enzyme reaction with respect to pH, ionic strength and divalent cations were demonstrated. The enzyme was most active at pH 8.5-9.0. Optimal concentration was 25 mM for (NHJ.SO,, 17 mM for Mg1+, and 0.6 mM for Mn1+. Properties of the Purified Enzyme—The molecular weight of the enzyme was calculated to be about 36,000 by Sephadex G-200 gel filtration (Fig. 7). We investigated effects of various nucleic acid on the ribonuclease H reaction (Table III). Single and double-stranded DNA showed very strong inhibition, while polyribonucleotides exhibited only slight inhibition. Mode of Action of Ribonuclease H—To elucidate the mode of action, ["QRNA-DNA hybrid was digested to various extents and the reaction products were chromatographed under conditions
F. TASHIRO and Y. UENO
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10 0 25 JO 100 200 Concnof (NH^)2SOA (mM)
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200 10
Molecular weight 100
0 10 20 0 1 2 3 A Concn of MgCl2 (mM) Coocn of MnCl2 (mM)
5
Fig. 6. Optimal concentrations for ribonuclease H activity. The experiments were earned out under the standard assay conditions with various pHs and concentrations of (NH4),SO4, MgCl,, and MnCl2. (A) Optimal pH of the enzyme. Tris-HCl buffer (50 mM) was used from pH 7.5 to 9.0, glycine-NaOH buffer (50 mM) from pH 9.0 to 10.0; (B) effect of ionic strength on the enzyme; ( Q dependency of the enzyme on MgCl t ; (D) dependency of the enzyme on MnCl,. Amount of enzyme used in experiments (A), (B), (C), and (D) was 3.5, 4 8, 1.6, and 1.6 units, respectively.
under which only small oligonucleotides would migrate (9, 17). At various stages of digestion by the enzyme, the bulk of the products remained as large molecules (Fig. 8, A-C). Even when 63% of the RNA moiety had been rendered acid-soluble, the amount of UMP was found to be very small. The data obviously indicate that the enzyme is an endonuclease. The resulting oligonucleotides were completely converted into 5'-UMP by snake venom phosphodiesterase (Fig. 8, D). The results strongly suggest that the oligonucleotides produced by the ribonuclease H of rat liver nuclei terminate in a 3'-hydroxyl group.
Fig. 7. Molecular weight of ribonuclease H determined by gel filtration. A published procedure was essentially followed (33). The column was 2x58 cm; the gel was Sephadex G-200; the elution buffer 0.5 M NH4C1 in buffer A. All operations were carried out in the manner described under Fig. 2. Myoglobin, chymotrypsinogen, ovalbumin, bovine serum albumin and ^-globulin (Schwarz/Mann) were detected by absorbance at 280 nm. Rat liver nuclear nbonuclease H was detected by activity measurement. Vo represents the void volume (estimated with blue dextran). Ke represents the elution volume.
TABLE HI. Effects of nucleic acids on ribonuclease H of rat liver nuclei. To the enzyme preincubated with each nucleic acid for 3 min at 37°C was added P4C]RNA-DNA hybrid and the mixture was incubated for 10 min at 37°C. The reaction was stopped by addition of 200 fi\ of 15% TCA and 100 y\ of yeast RNA (2 mg/ml) and the acid-soluble radioactivity was assayed as described in " MATERIALS AND METHODS." Amount of enzyme used was 6.7 units. Activity ([% of control) 12.5
/ig/assay 25 50 100
87
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Poly(rA)
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93
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Poly(rC)
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3
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Calf thymus DNA (denatured)
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J. Biochem.
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NUCLEAR RIBONUCLEASE H FROM RAT LIVER
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Fig. 8. Chromatography of digestion products of rat liver nuclear ribonuclease H. ["QRNA-DNA hybrid (3,897 cpm) with labelled UMP residues was incubated at 37°C for various lengths of time with the enzyme (43.3 units). The extent of degradation, determined by the standard assay, is indicated in parentheses. The reaction mixture was subjected to descending paper chromatography in the manner described previously (9). One-centimeter strips were cut from the chromatogram and assayed for radioactivity. O represents the origin, pU the position of 5'-UMP, and U the position of uridine. A) Unreacted hybrid, B) digestion for 7 min, Q digestion for 15 min, D) digestion for 15 min followed by treatment of half of sample with snake venom phosphodiesterase for 30 min at 37°C.
20 30 Fraction number
Fig. 9. DEAE-Sephadex chromatography of nucleoplasmic and nucleolar ribonuclease H and RNA polymerase. The crude extract was applied to the DEAESephadex A-25 column in the manner described under Fig. 1. , (NH4),SO4 concentration; O, ribonuclease H activity; • , RNA polymerase activity. A) The crude extract from nucleoplasmic fraction, B) the crude extract from nucleolar fraction.
The RNA polymerase activity eluting at 0.15 M (NH4),SO4 on DEAE-Sephadex chromatography was completely insensitive to a-amanitin (1 /ig/ml), while the RNA polymerase activity eluting at 0.25 M (NH4),SO4 was completely sensitive to low concentration of a-amanitin (1 /^g/ml). We concluded that the former enzyme was RNA polymerase I and the latter RNA polymerase II (18). The Intranuclear Localization of the Enzyme—To preparation of nucleoplasm hardly exhibited any investigate the localization of the enzyme in the RNA polymerase I activity in all experiments. nucleus, we extracted the enzymes from the nucle- Although the preparation of nucleoli contained oplasm and nucleoli and compared their properties. some RNA polymerase II activity (Fig. 9), the It is well known that DNA-dependent RNA large amount of ribonuclease H activity contained polymerases I and II exist in nucleoli and nucle- in the preparation of nucleoli could not be acoplasm, respectively. Therefore, we checked the counted for by contamination by the nucleoplasm. purity of our preparations of nucleoli and nucleThe two ribonuclease H preparations from the oplasm on the basis of their contents of these two nucleoli and nucleoplasm could not be distinguished RNA polymerase activities. from each other on the basis of such properties as Vol. 84, No. 2, 1978
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DISCUSSION We have partially purified and characterized the nuclear ribonuclease H of rat liver. As summarized in Table I, the purification with phosphocellulose chromatography resulted in an inexplicable loss of the enzyme activity. The introduction of more effective purification procedures such as DNA-cellulose chromatography is expected to increase the yield of the enzyme activity. The properties of rat liver nuclear ribonuclease H were distinct from those of rat liver cytosol enzyme reported by Roewekamp and Sekeris (10) in many respects, such as molecular weight, optimal pH and requirements for Mgs+ and Mn'+ ions. As reported in the accompanying paper, we prepared the rat liver cytosol enzyme by the method of Roewekamp and Sekeris (10) and confirmed these differences between the nuclear and cytosol enzymes. Multiple forms of ribonuclease H have been reported in various eukaryotic organisms (1-9). These reports and our present results imply that multiple forms of ribonuclease H are present in different regions of the cell in higher organisms. As for its substrate specificity, the nuclear enzyme rapidly hydrolyzed up to 65 % of the RNA moiety of RNA-DNA hybrid but not the RNA dissociated from the hybrid by heat denaturation. Degradation of single or double-stranded DNA, poly(rU) and poly(rU)-poly(rA) was negligibly low. Poly(rU)-poly(dA) showed a poor substrate activity. In the presence of Mg1+ ions, the nuclear enzyme was very similar in its action on poly(rU)poly(dA) to the ribonuclease H isolated from rat liver cytosol (10), Ustilago maydis (3), human leukemic blood cells (19), and calf thymus (4), but different from that of KB-cells (20). Analysis of the reaction products demonstrated that the enzyme cleaves the RNA chain endonucleolytically producing oligonucleotides with 3'-hydroxyl termini. This mode of action is similar to that
of ribonuclease H of other cellular origins (2, 10, 14, 20-22). In the presence of polyribonucelotides the enzyme activity was slightly inhibited, but the inhibition was complete in the presence of single or double-stranded DNA. The same results were also obtained in the case of Raucscher leukemia virus ribonuclease H (23). These results strongly suggest that ribonuclease H first binds to the DNA region of RNA-DNA hybrid, recognizes the RNA of the hybrid and finally degrades the RNA region. When RNA-DNA hybrid used as substrate is contaminated with free RNA or single-stranded DNA and the enzyme preparation contains other nucleases, the exact determination of ribonuclease H activity is very difficult. For this purpose it is preferable to use the RNA-DNA hybrid treated with SI nuclease (34). The existence of multiple forms of ribonuclease H in rat liver and other organisms raises the question of the biological function of the enzymes as well as of their substrate, the RNA-DNA hybrid. The demonstration of ribonuclease H in the nucleus strongly corroborates the possible role of this enzyme in the metabolic process of nucleic acid. Several authors have indicated that ribonuclease H might be involved in the removal of the initiator RNA chain priming DNA synthesis (24-28), while others have suggested a possible role in the process of transcription (7, 29-31). Additionally, Wyers et at. have postulated a possible role in the chromatin structure (6). The existence of only one species of nuclear ribonuclease H suggests that the same kind of ribonuclease H functions in both ribosomal RNA and heterogeneous nuclear RNA synthesis although different forms of DNA-dependent RNA polymerase are involved in the respective processes. Blisen et al. have reported that ribonuclease H lib increased in parallel with RNA synthesis and ribonuclease H I increased together with DNA synthesis during the response of bovine lymphocytes to concanavalin A (32). It is still premature to define the real role of ribonuclease H. However these observations point to the interesting possibility that ribonuclease H plays a crucial role in the metabolism of nucleic acid.
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optimal concentrations of divalent cations, sensitivity to rifampicin derivatives, behavior on DEAESephadex and phosphocellulose column chromatography. From these results, we infer that the ribonuclease H of rat liver nuclei is only one species and exists in both nucleoplasm and nucleoli.
NUCLEAR RIBONUCLEASE H FROM RAT LIVER
REFERENCES 1. Sekeris, C.E. & Roewekamp, W. (1972) FEBS Lett. 23, 34-36 2. Banks, G.R. (1974) Eur. J. Biochem. 47, 499-507 3. O'Cuinn, G., Persico, F.J., & Gottlieb, A.A. (1973) Biochim. Biophys. Ada 324, 78-85 4. BUsen, W. & Hausen, P. (1975) Eur. J. Biochem. 52, 179-190 5. Wyers, F., Sentenac, A., & Fromageot, P. (1976) Eur. J. Biochem. 69, 377-383 6. Wyers, F., Huet, J., Sentenac, A., & Fromageot, P. (1976) Eur. J. Biochem. 69, 385-395 7. Naton, S., Takeuchi, K., Takahashi, K., & Mizuno, D. (1973) /. Biochem. 73, 879-888 8 Cooper, R.J., Duff, P.M., Oliver, A., Craig, R.K., & Keir, H.M. (1974) FEBS Lett. 45, 38-^3 9. Tashiro, F., Mita, T., & Higashinakagawa, T (1976) Eur. J. Biochem. 65, 123-130 10. Roewekamp, W. & Sekeris, C.E. (1974) Eur. J. Biochem. 43, 405-413 11. Davison, P.F. & Freifelder, D. (1962) /. Mol. Bwl. 5, 635-642 12. Roeder, R.G. & Rutter, W.J. (1970) Proc. Natl. Acad. Sci. U.S. 65, 675-682 13. Higashinakagawa, T., Tashiro, F., & Mita, T. (1975) J. Biochem. TJ, 783-793 14. Harberkern, R.C. & Cantoni, G.L. (1973) Biochemistry 12, 2389-2395 15. Bray, G.A. (1960) Anal. Biochem. 1, 279-285 16. Higashinakagawa, T., Muramatsu, M., & Sugano, H. (1972) Exptl. Cell Res. 71, 65-74
Vol. 84, No. 2, 1978
17. Lapidot, Y. & Khorana, H.G. (1963) J. Am. Chem. Soc. 85, 3857-3862 18. Weimann, R. & Roeder, R.G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1790-1794 19. Sargadharan, M.G., Leis, J.P., & Gallo, R.C. (1975) /. Biol. Chem. 250, 365-373 20. Keller, W. & Crouch, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3360-3364 21. Leis, J.P., Bcrkower, I., & Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U.S. 70, 466-470 22. Miller, H.I., Riggs, A.D., & Gill, G.N. (1973) / . Biol. Chem 248, 2621-2624 23. Modak, M.J. & Marcus, S.L. (1977) / . Virol. 22, 243-246 24. Wickner, W., Brutlag, D., Schekman, R., & Komberg, A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 965-969 25. Lark, K.G. (1972) J. Mol. Biol. 65, 47-60 26. Sugino, A., Hirose, S., & Okazaki, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1863-1867 27. Keller, W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1560-1564 28. Spadari, S. & Weissbach, A. (1975) Proc. Natl. Acad. Sci. U.S. 72, 503-507 29. Sekeris, C.E., Schmid, W., & Roewekamp, W. (1972) FEBS Lett. 24, 27-31 30. Huet, J., Wyers, F., Buhler, J.M., Sentenac, A., & Fromageot, P. (1976) Nature 261, 431-433 31. Doenecke, D., Marmaras, V.J., & Sekeris, C.E. (1972) FEBS Lett. 11, 261-264 32. BUsen, W., Peters, J.H., & Hausen, P. (1977) Eur. J. Biochem. 74, 203-208 33. Andrews, P. (1964) Biochem. J. 91, 222-233 34. Sutton, W.D. (1971) Biochim. Biophys. Ada 240, 522-531 35. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) /. Biol. Chem. 193, 265-275
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We wish to thank Dr. T. Mita of National Cancer Center Research Institute and Dr. T. Higashinakagawa of Mitsubishi-Kasei Institute of Life Sciences for helpful discussions and Drs. T. Ando and E. Hayase of The Institute of Physical and Chemical Research for preparation of 'H-Iabelled T7 DNA.
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