Two ribonuclease H activities from the murine myeloma, MOPC-21

Biochimica et Biophysica Acta, 324 (1973) 78-85 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97800

TWO RIBONUCLEASE H ACTIVITIES FROM THE MURINE MYELOMA, MOPC-21

GERARD O'CUINN*, FRANCIS J. PERSICO** and A. ARTHUR GOTTLIEB*** Institute of Microbiology, Rutgers University, New Brunswick, N. J. 08903 (U.S.A.) (Received May 21st, 1973)

SUMMARY Two distinct enzymatic activities capable of degrading the R N A strand of an R N A • D N A hybrid have been isolated from the murine myeloma, MOPC-21. These activities can be distinguished by their respective chromatographic and sedimentation properties as well as p H optima and divalent cation requirements.

INTRODUCTION An enzyme, capable of degrading the R N A strand of a D N A • R N A hybrid, was first isolated by Stein and Hausen l'z from calf thymus. Recently similar activities have been described in Escheriehia coli, avian myeloblastosis virus and other animal cells 3-7. Since R N A can serve as a primer for D N A synthesis 8, it has been suggested that ribonuclease H may play an important role in the control of D N A synthesis by virtue of its ability to selectively remove initiating R N A strands. The murine myeloma is a particularly interesting type of malignant cell, since it retains a high degree of differentiation with respect to the production of homogeneous ?-globulin. In view of the observation of Koros etaL 9 that antibody-forming cells contain large amounts of R N A • D N A hybrid, and the possibility that the R N A components of such hybrids might play an important role in the control of D N A synthesis in this cell line, it seemed reasonable to examine this line for ribonuclease H activity. In this report, we describe the isolation of two such activities which can be distinguished on the basis of differences in sedimentation and chromatographic behavior as well as p H optima and divalent cation requirements. METHODS Extraction and pur(fication o f ribonuclease H activity f r o m mouse myeloma tissue The myeloma line MOPC-21 was maintained by serial passage in Balb/c mice.

* Present address: Department of Biochemistry, University College, Galway, Ireland. ** Present address: Ortho Research Foundation, Raritan, New Jersey. *** To whom reprint requests should be addressed.

TWO RIBONUCLEASE H ACTIVITIES

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For the extraction of ribonuclease H activity, the method of Stein and Hausen 1 was employed. A crude extract was prepared by disrupting the tissue, using an omnimixer, in a volume of 0.01 M Tris-HCl buffer (pH 7.8)-15 mM 2-mercaptoethanol equivalent to four times the tissue weight. This crude extract was then centrifuged at 27 000 × g for 30 min and the pellet discarded. To the resultant high supernatant fraction, (NH4)2SO 4 and MnC12 were added to final concentrations of 0.05 M and 2 mM, respectively. The supernatant was then added to a slurry of DEAE-cellulose (Serva) previously equilibrated with buffer containing 0.01 M Tris-HC1 (pH 7.8), 15 m M 2-mercaptoethanol, 0.05 M (NH4)2SO4 and 2 mM MnC12. The mixture was stirred for 25 rain and then spun at 3000 × g for 20 min to remove the DEAE-cellulose. The resultant supernatant (Fraction A) was diluted with 30°.~ glycerol, 0.01 M Tris-HCl (pH 7.8), 15 mM 2-mercaptoethanol and 2 mM MnC12, until the ionic strength was reduced to an ionic strength less than 0.02 M KC1 in this buffer. After loading of this fraction onto a 1 c m × 25 cm DEAE-cellulose (Whatman) column and washing of the column with Tris-glycerol-mercaptoethanol-MnC12 buffer, 0.05 M KCl in Tris-glycerol-mercaptoethanol-MnC12 buffer was applied to the column. Elution was continued until the A28o nm of the eluant was less than 0.02 A units. Fractions containing enzyme activity in the flow-through and 0.05 M eluate were collected, pooled and separately diluted in Tris-glycerol-mercaptoethanol-MnCl 2 buffer to an ionic strength of less than 0.02 M KC! in buffer. Application of 0.3 M KC1 (in this buffer) to the DEAE-cellulose column at that point resulted in no further release of ribonuclease H activity from the column. After adjustment of the pH of the flow-through and 0.05 M eluate to 7.0, the fractions were loaded onto separate phosphocellulose columns of similar size (1 crux 15 cm) previously equilibrated with Tris-glycerol-mercaptoethanol-MnCl 2 buffer (pH 7.0). If this procedure was carried out in the absence of Mn 2+, the elution characteristics of H-1 and H-2 were similar, but the yield was somewhat less than that achieved in the presence of Mn 2 +. After washing of the columns with Tris-glycerol-mercaptoethanol-MnCl 2 buffer, a linear gradient of KC1 (0 to 0.5 M) in buffer was applied, 2-ml fractions were collected and aliquots were assayed for enzyme activity. Assay o f ribonuclease H activities Preparation o f R N A • D N A hybrid. 48 mg of calf thymus DNA was dissolved overnight in 0.01 M Tris-HC1 buffer (pH 7.8) containing 0.01 M NaC1 and treated with 100/~g/ml ribonuclease A at 37 °C for 30 rain. Self-digested pronase (1 mg/ml) was then added, and the mixture incubated for another 30 rain at 37 °C. The ribonuclease and pronase were removed from this mixture for two cycles of extraction with phenol, and the D N A was precipitated with 2 vol. of ethanol, and collected by sedimentation. The D N A was dissolved in 0.01 M Tris-HCl buffer containing 1 mM NaC1 (pH 7.8) and dialyzed extensively against this buffer. Denaturation of the D N A was accomplished by heating the D N A in 0.01 M Tris-HCl-1 mM NaC1 (pH 7.8) for 5 min at 90 °C followed by quick cooling. 900/~g of denatured D N A was used as template for E. coli DNA-dependent R N A polymerase (spec. act. 1200 units/mg) in a reaction mixture containing 0.05 M Tris-HC1 buffer (pH 7.8), 160 mM KCI, 1 2 m M Mg z+, 1 2 m M 2-mercaptoethanol, 5 mg/ml bovine serum albumin, 3.45 /~moles of G T P and CTP, 2.19 pmoles of ATP, and 0.41/tmoles of UTP. [3H]UTP was present at a spec. act. of 375 cpm/pmole. Incubation was carried out at 37 °C

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G. O'CUINN et al.

for 60 min and the reaction was stopped by addition of sodium dodecyl sulfate to a final concentration of 10 %. The mixture was then passed over a Sephadex G-25 column to separate the labelled hybrid from labelled precursor and the detergent. The hybrid was pooled, precipitated by addition of 3 vol. of ethanol, and collected by sedimentation. The precipitated hybrid was resuspended in 0.01 M Tris-HCI (pH 7.8)-0.1 M NaC1 and dialyzed overnight against the same buffer. Reaction mixture f o r ribonuclease H assay Ribonuclease H activity was assayed by scoring for release of [3H]uridylate from 145 ng of R N A • D N A hybrid containing 8800 cpm of labelled uridylate in the R N A strand. In general, assays were carried out in volumes of 0.5 ml containing 0.05 M Tris-HC1 (pH 7.8), 2.0 mM Mn 2÷, 0.01 M (NH4)2SO 4, and 50/al of enzyme fraction to be tested. After 20 min of incubation at 37 °C, each reaction mixture was treated with 0.5 ml of ice-cold 20 % trichloroacetic acid containing 200/~g of carrier low mol. wt RNA. The mixtures were then filtered on 0.45-#m Bac-T-Flex (Schleicher and Schuell) filters and the filtrate was counted in a 33 % Triton X-100-0.6 % P P O 0.04 % POPOP-toluene scintillation mixture. All assays were carried out under conditions in which the release of [3H]uridine was linearly proportional to the amount of each fraction added. Background values were obtained by substituting 50/~1 buffer [50 mM Tris-HC1 (pH 7.8), 12.5 mM 2-mercaptoethanol, 20 % glycerol and 1 mg/ml bovine serum albumin] for 50 l~l of enzyme in the assay mixture. For assays in which cofactor or H ÷ variations were employed, the conditions are given in the legends to the appropriate figures.

RESULTS A cell-free extract of myeloma cells from the MOPC-21 contains activities which degrade the R N A moiety of an R N A • D N A hybrid. This activity can be separated into two fractions by chromatography on DEAE-cellulose (Fig. 1). The enzyme 8(3

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Fig. 1. glution of ribonuclease H activities from DEAg-cellulose. Fraction A was diluted to a conductivity less than 0.02 M KC1 in Tris-glycerol-mercaptoethanol-MnCl2 buffer and applied to a Whatman DEAE-cellulose column. The column was washed with Tris-glycerol-mercaptoethanolMnCI2 (pH 7.8). 50 #1 of the fractions indicated were assayed for ribonuclease H activity as described in Methods. The 0.05 M cut was commenced at Fraction 52.

TWO RIBONUCLEASE

H ACTIVITIES

81

which does not adsorb to DEAE-cellulose has been termed H-1 to distinguish it from the enzyme which elutes at 0.05 M KCI (H-2). After recovery from DEAE-cellulose, both H-1 and H-2 display appropriate elution behavior upon rechromatography on DEAE-cellulose. Fig. 2 displays the elution characteristics of H-1 and H-2 during phosphocellulose chromatography. It is apparent that the H-1 activity binds more I

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Fig. 2. Elution profile on phosphocellulose of the ribonuclease H activities recovered by D E A E cellulose chromatography. Both e n z y m e s recovered from the DEAE-cellulose column shown in Fig. 1 were applied separately to columns of phosphocellulose. Following extensive washing of these columns with Tris-glycerol-mercaptoethanol-MnCl2 buffer (pH 7.0), elution of these activities was carried out using a linear gradient of KC1 (0.0 to 0.5 M). In this figure, the gradients for the two phosphocellulose columns were coincident and the distinct elution profile of each enzyme is shown. 50 ~1 aliquots of each fraction were assayed for ribonuclease H activity. 5

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Fig. 3. Sensitivity of native and denatured hybrid to ribonuclease H activity. The H-1 and H-2 fractions were tested against native and heat denatured R N A . D N A hybrid prepared as described in Methods. In each case, native or denatured hybrid containing 8800 c p m of [3H]ribouridylic acid was added to assay mixtures containing 50-/~1 aliquots fractions o f H-1 (0.22/~g total protein) and H-2 (0.26 # g total protein), x -- x and x - - - x refer to the action of Fraction H-1 on native and denatured hybrid, respectively, while O - O and O - - - O refer to the action of Fraction H-2 on these substrates. Each point represents the average of duplicate determinations.

G. O ' C U I N N et al.

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tightly to phosphocellulose than does H-2. H-1 and H-2 are therefore distinguishable in two different chromatographic systems. Fig. 3 demonstrates that both enzymes exhibit preference for R N A in the R N A . D N A hybrid as compared with R N A in single-stranded form released from the hybrid by denaturation. The activities of both ribonuclease H activities displayed linearity with respect to protein concentration and as shown in Fig. 3, the activities were linear with respect to time for up to 180 min. Table I presents the relative yield and degree of purification achieved for the two ribonuclease H activities. On the basis of the specific activity of the initial cell TABLE I PURIFICATION

OF R I B O N U C L E A S E

H ACTIVITIES FROM MYELOMA TISSUE

Fraction

Total ing protein

Total activity (~moles)*

Spec. act. (pmoles/t~g)

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278.2 113.2 34.4 22.2 4.6 1.1 0.24

33.9 19.3 12.2 19.9 3.9 3.3 0.81

121.8 170.5 356.6 894.9 854.9 3032.4 3425.0

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Fig. 4. The effect of pH on the activity of the ribonuclease I-1 fractions. 50-/~1 aliquots of the H-1 and H-2 fractions contain 0.55/~g and 0.65/xg protein, respective/y, were assayed as described in Methods under conditions of variable pH. Fraction H-1 was incubated with the hybrid for 3 h in the presence of 2.0 M Mn 2+, while Fraction H-2 was incubated with hybrid for 1 h in the presence of 0.6 m M Mn 2+.

TWO RIBONUCLEASE H ACTIVITIES

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Fig. 5. Mg 2+ and Mn 2+ optima for activity of Fractions H-I and H-2. 50-bd aliquots of the H-1 and H-2 fractions containing 0.55/~g and 0.65 # g protein, respectively, were assayed as described in Methods. Assays employing Fraction H-2 were carried out for 20 min using Mn 2 ÷ and for 2 h using Mg 2+. The results are displayed in Panel A. All assays employing fraction H-1 were carried out for 3 h and are s h o w n in Panel B.

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Fig. 6. Glycerol gradient sedimentation o f Fractions H-I and H-2. Aliquots o f H-1 and H-2 each containing 11/~g o f protein were separately layered over 10-40 ~g glycerol gradients, and centrifuged in an SW 40 Rotor at 36 000 rev./min for 32 h at 4 °C. Fractions were collected by puncturing the tubes and 50-/~1 aliquots of each fraction were assayed for ribonuclease H activity.

free extract, the H-2 enzyme was purified approximately 25-fold while the H-1 enzyme was purified 28-fold.

84

G. O'CU1NN et al.

The two ribonuclease H activities could also be distinguished from each other on the basis of p H optima and cofactor requirements. Fig. 4 illustrates the activity of each enzyme as a function of p H while Fig. 5 demonstrates the differences in Mg 2+ and Mn z+ optima for each enzyme. Sedimentation analysis of the H-1 and H-2 activities revealed that H-1 consistently sedimented faster than H-2 (Fig. 6). When run in parallel with catalase as a standard, the sedimentation coefficient of H-1 was 7.0-8.0 and that of H-2 was 4.0-5.0. DISCUSSION Our studies have shown that two enzymes of the ribonuclease H type can be recovered from the MOPC-21 murine myeloma line. The two enzymes are distinguishable by physical and biochemical criteria. Following the report by Stein and Hausen I that such an enzyme is present in calf thymus, and the demonstration that R N A strands may serve as initiators for D N A synthesis, it became clear that such an enzyme may play a critical role in the control of D N A synthesis by digesting R N A initiator strands. Indeed, the description by Koros e t al. 9 of large amounts of R N A • D N A hybrid in antibody forming cells is of interest in this regard. Enzymes of this type have been isolated from the avian myeloblastosis virus 5'6, from E . coli 6 and from several eukaryotic cells 7. The enzyme from avian myeloblastosis virus appears to be an exonuclease, while those from E. eoli and animal cells appear to be an endonuclease. The present report is the first description of the isolation of more than one of these enzymes from a malignant line of cells. Since the myeloma cell contains C-type viral particles 1° it has been suggested that one or both of these activities might arise from such C-type particles. We think this is unlikely, since the ribonuclease H activity in the avian myeloblastosis virus cannot be dissociated from the D N A polymerase of this virus. In contrast, neither the H-1 or H-2 activities from the myeloma exhibit D N A polymerase activity as tested on a variety of natural and synthetic nucleotide templates, and none of the multiple D N A polymerase enzymes which we have isolated from this cell 11,12 contain ribonuclease H activity. It is not known whether the A particle 13 enzyme described by Wilson and Kuff 14 contains either of our ribonuclease H activities. Recently, Sekeris and Roewekamp 4 have observed two ribonuclease H activities following salt elution gradient chromatography on phosphocellulose of material derived from a rat liver extract 4. The relationship of these activities to the myeloma activities described in this report remains to be determined. Our method provides a way by which these activities can be recovered from the myeloma cell for in vitro study and characterization. ACKNOWLEDGMENTS This study was supported by grants from the American Cancer Society (NP79B), the D a m o n Runyon Fund (DRG-1213) and the National Science Foundation (GB-16871).

TWO R I B O N U C L E A S E H ACTIVITIES

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REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Stein, H. and Hausen, P. (1969) Science 166, 393 Hausen, P. and Stein, H. (1970) Eur. J. Biochem. 14, 278 Keller, W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1560 Sekeris, C. E. and Roewekamp, W. (1972) F E B S Lett. 23, 34 Molling, K., Bolognesi, D. P., Bauer, H., Busen, W., Plassman, H. W. and Hausen, P. (1971) Nat. N e w Biol. 234, 240 Leis, J. P., Berkower, I. and Hurwitz, J. (1973) Proc. Natl. Acad. Sci. U.S. 70, 466 Keller, W. and Crouch, R. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3360 Brutlag, D., Schekman, R. and Kornberg, A. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2826 Koros, A. M., Koster, L. H. and Mowery, M. J. (1971) Nat. N e w Biol. 232, 239 Watson, F., Ralph, P., Sarkar, S. and Cohn, M. (1970) Proc. Natl. Acad. Sci. U.S. 66, 344 Persico, F. J. and Gottlieb, A. A. (1972) Nat. New Biol. 239, 173 Persico, F. J., Nicholson, D. E. and Gottlieb, A. A. (1973) Cancer Res. 33, 1210 Kuff, E. L., Leuders, K. K., Ozer, H. L. and Wivel, N. A. (1972) Proc. Natl. Acad. Sci. U.S. 69, 218 Wilson, S. H. and Kuff, E. L. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1531