RNAase-sensitive DNA-dependent DNA polymerase from rat cells transformed by avian sarcoma virus

92

Biochimica et Biophysica A cta, 781 (l 984) 92-99

Elsevier BBA 91319

R N A a s e - S E N S I T I V E DNA-DEPENDENT DNA P O L Y M E R A S E F R O M RAT C E L L S T R A N S F O R M E D BY AVIAN S A R C O M A V I R U S MOSHE KOTLER a, j. ROSENBERG a and BERTOLD FRIDLENDER t, a Department of Molecular Virology, Hebrew University -Hadassah Medical School, 91 010 Jerusalem and h International Genetic Sciences Partnership, P.O.B. 4330, Jerusalem (Israel)

(Received June 24th, 1983)

Key words: DNA polymerase," RNAase sensitivity," Avian sarcoma virus," (Rat fibroblast)

An RNAase-sensitive DNA polymerase from rat cells transformed by avian sarcoma virus has been characterized. The enzyme requires RNA for its activity, as shown by its sensitivity to RNAase with endogenous as well as exogenous DNA templates. This sensitivity is maintained after its purification by sucrose gradients and ion exchange columns. A molecular weight of about 100000 has been estimated. This DNA polymerase requires high salt concentration for its activity, is resistant to high concentrations of phosphonoacetic acid (400 /tg/ml), is partially inhibited by 5 mM N-ethylmaleimide, and is completely inhibited by 0.3 mM parahydroxymercuribenzoate.

Introduction Eukaryotic cells have been found to contain several D N A polymerase activities designated: c~, fl, "t and 8 [1-5], as well as RNAase-sensitive D N A polymerases [6-15]. Two possible functions have been proposed for the role of R N A in the process of D N A replication. One function is to serve as a template for the synthesis of D N A [10,11,15] in a way similar to the reverse transcriptase activity. The other is to act as primer for the growth of D N A chains [16,17]. As shown by Spadari and Weissbach [18], only D N A polymerase a was able to synthesize D N A covalently linked to R N A when a H e L a cell D N A hybridized to a short R N A primer molecule was used as template. However, a clear-cut role for R N A in D N A synthesis has not been established. We have previously demonstrated [12] an RNAase-sensitive D N A polymerase activity in rat cells transformed by the B77 strain of avian sarcoma virus. This enzyme differs from the reverse transcriptase by its requirement of D N A as 0167-4781/84/$03.00 © 1984 Elsevier Science Publishers B.V.

template, its ability to act on synthetic templates, and its sensitivity to actinomycin D. Similar activities have been found in phytohemagglutininstimulated lymphocytes [13], human embryo cells [19], allantoic fluid of uninfected leukosis-free eggs [16] and chick embryos [10]. Here we present some of the properties of the partially purified D N A polymerase and show that the RNAase sensitivity of this enzyme is due to the requirement of R N A molecules for the DNA-dependent D N A polymerase activity.

Methods Cells. The RB77 cell line of rat embryo fibroblasts were transformed in vitro by the B77 strain of avian sarcoma virus [20]. Cells were cultured in RPMI-1640 medium (Grand Island Biological Co.), supplemented with 10% tryptose phosphate broth (Difco) and calf serum. 32p L a b e l i n g . After incubation for 3 h in phosphate-deficient RPMI-1640 medium, the cells in three Roux bottles were fed with RPMI-1640 con-

93 taining 100 /~Ci/ml of [32p]orthophosphate (Nuclear Research Center, Negev, Israel). After 78 h incubation at 37°C, the ceils were harvested and the enzyme was extracted. DNA polymerase assay. The assay measures the incorporation of [3H]dTMP into an acid-insoluble product. The polymerase assay was carried out in a standard mixture containing 10 mM Tris-HC1, p H 8.3/10 mM MgC12/40 mM KC1/0.125 mM each of dATP, dGTP, d C T P / 0 . 2 0 mM [3H]dTTP (Amersham, U.K.). 1 enzyme unit is defined by the incorporation of 1 pmol dTTP in 1 min and was equivalent to 500 cpm in most of the experiments. Exogenous reactions were carried out in the presence of 8 0 / ~ g / m l activated calf thymus DNA [21]. The enzyme preparations were incubated for 10 min at 37°C with or without RNAase (20 /~g/ml) (Sigma), followed by addition of the assay mixture. The RNAase was incubated at 80°C for 3 min before use. The reactions were stopped by transferring samples of 10 or 20 /~1 into cold trichloroacetic acid. Purification of the enzyme. The initial purification of the enzyme followed the procedures previously described [12,15]. In brief, the cell pellet was suspended in 10 ml buffer A (0.01 M Tris-HC1, p H 7.8/0.1 M NaCI/0.001 M EDTA) containing 0.25 M sucrose, 0.03 M dithiothreitol and 0.25% ( v / v ) Nonidet P-40 and suspended by gentle pipetting. This was followed by centrifugation for 10 min at 10000 rpm in an SW 50.1 Beckman rotor at 4°C. The supernatant fluid was centrifuged for 60 min at 45 000 rpm in the same rotor. The pellet resulting from this centrifugation was suspended in 1 ml buffer B (0.1 M Tris-HCl, pH 7.8/0.001 M EDTA) containing 0.03 M dithiothreitol and will be referred to as the high-speed pellet. To the resuspended high-speed pellet, ammonium sulfate (0.436 g / m l ) was added and the solution was kept in ice for 30 min and then centrifuged at 45 000 rpm for 20 min at 4°C in a Beckman 50 Ti rotor. The pellet was resuspended in 1-2 ml buffer B containing 1 mM dithiothreitol and dialyzed against 2 1 of the same buffer for 12-16 h. The extract was then used for further purification by sucrose gradient centrifugation or ion exchange chromatography. Purification on sucrose gradients. The dialyzed extracts were layered onto a 5-20% (w/v) continu-

ous sucrose gradient prepared in buffer B containing 1 mM dithiothreitol and 10 mM KCI and centrifuged for 15 h at 35000 rpm in an SW 50.1 Beckman rotor. The gradients were fractionated dropwise and 10 /~1 aliquots were assayed using activated DNA as a template. Ion exchange chromatography. 5 ml phosphocellulose or 5 ml DEAE-cellulose (DE-52) (Whatman) were equilibrated with buffer B containing 20% glycerol and 10 mM fl-mercaptoethanol. The dialyzed DNA polymerase preparations containing 0.1-1 mg protein in 0.1-1 ml were applied to the column (built in a 10 ml plastic pipet) and washed with 3 bed vol. of buffer B, the enzymes were eluted, using a 0-0.5 M KC1 gradient in buffer B. Fractions were collected into tubes containing 100/tg bovine serum albumin and aliquots of 5 /~1 from each fraction were used to assay enzymatic activity. Results

Purification of RNAase sensitive DNA polymerase The kinetics of incorporation of [3H]dTMP by the enzyme present in the high-speed pellet are seen in Fig. 1. The enzymatic fractions were incubated with or without activated DNA. A high activity was observed when exogenous template was added to the reaction mixture. Polymerase activity on both endogenous and exogenous template was completely inhibited by pretreatment of the extract with RNAase. These results demonstrated that the RNAase sensitivity of the enzyme remains unaltered even in the presence of exogenous primer template DNA, suggesting that the R N A component is not needed as primer. The following experiment was carried out to show that the sensitivity of this DNA polymerase is due to the RNAase activity and not to the presence of a basic protein (RNAase) in the reaction mixture. Pancreatic RNAase was inactivated with diethyl pyrocarbonate. The diethyl pyrocarbonate was removed by raising the temperature to 40°C for 10 rain and incubating under vacuum for 1 h at room temperature. As shown in Table I, the inactivated RNAase had no effect on the DNA polymerase activity, while RNAase incubated under the same conditions without diethyl pyrocarbonate reduced the DNA polymerase activity

94

In support of this conclusion, it was observed that when a high-speed pellet is passed at 37°C through a column of RNAase bound to agarose (ENzite-Agarose Ribonuclease, Miles Laboratories), the activity of the enzyme in the eluate is reduced to 20% of the input, without change in the protein concentration. No RNAase activity was eluted with the enzyme (data not shown). The high-speed pellets were further purified by precipitation with ammonium sulfate, followed by centrifugation on sucrose gradients. It was found (Fig. 2A) that the enzyme sedimented in the sucrose gradient as a sharp band and its activity was still sensitive to RNAase. The various steps in the purification of the enzyme are summarized in Table II. The sucrose gradient centrifugation, resuited in a 1300-fold purification. The activity of the RNAase-sensitive DNA polymerase in the crude extracts was lower than in the subsequent purification steps, probably because of the presence of RNAase or other inhibitors in the crude preparations. The endogenous activity of the enzyme in the absence of added template primer DNA, disappeared after ammonium sulfate precipitation and centrifugation in sucrose gradient (Fig. 2A insert). At this stage the activity was fully dependent on an exogenous template and was still fully sensitive to RNAase.

EXOGENOUS

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TIM[ (Bin) Fig. 1. Kinetics of [3H]dTMP incorporation. Kinetics of D N A synthesized by the high-speed pellet in the presence (A) or absence (e) of exogenous activated calf thymus DNA, and its sensitivity to RNAase (zx, O). The enzyme preparations were incubated for 10 min at 37°C with or without R N A a s e (20 # g / m l ) prior to the addition of the assay mixture.

to 5%. Thus, it can be concluded that RNAase activity by itself causes the inhibition of the D N A polymerase activity. TABLE I

T H E E F F E C T O F R N A a s e A N D I N A C T I V A T E D R N A a s e ON T H E D N A P O L Y M E R A S E P R E S E N T IN T H E H I G H - S P E E D PELLET 200 # g / m l pancreatic RNAase treated or untreated with diethyl pyrocarbonate a (bycovin) 40 # l / m l (Sigma), and 10 m M Tris solution (pH 8.3), treated or untreated with diethyl pyrocarbonate (controls), were incubated at 40°C for 10 min and then subjected to vacuum pressure for 1 h at room temperature to evaporate the diethyl pyrocarbonate. These solutions were mixed with 3H-labeled r R N A or with a high-speed pellet to a final concentration of 40 # g / m l RNAase and incubated for 10 min at 37°C. The radioactivity of the 3H-labeled r R N A was determined by trichloroacetic acid precipitation, and the D N A polymerase activity was determined in the presence of activated D N A after additional incubation of 20 min at 37°C in the presence of the polymerase reaction mixture, as described in Fig. 1. Treatment

3 H-labeled

rRNA

RNAase-sensitive D N A polymerase activity

cpm Control Control with bycovin a RNAase Inactivated R N A a s e with bycovin

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Fig. 2. Purification of RNAase-sensitive DNA polymerase extracted from unlabeled and 32P-labeled cells through sucrose gradients. A. The gradients were fractionated and aliquots of 10 pl were used to assay the enzyme activity in the presence of activated DNA without (e) or with (O) RNAase. The insert shows the kinetics of DNA synthesis by the enzyme in the peak fraction described in Fig. 2A without (e) or with (O) RNAase pretreatment, and without addition of activated DNA (A). B. A high-speed pellet from 32P-labeled cells was fractionated on sucrose gradients. Amounts of 32p radioactivity (*, zx), as well as DNA synthesis measured by [3H]dTMP incorporation (e, O), were determined in each fraction without (A e) and with (zx, O) pretreatment with RNAase. Molecular weight markers were run in parallel gradients; their position is indicated by arrows. IgG ('yglobulin); BSA (bovine serum albumin); OVA (ovalbumin); p27 (major protein of B77 virus). TABLE II PURIFICATION OF THE RNAase-SENSITIVE DNA POLYMERASE The enzyme was purified as described in Fig. 2. At each step, the enzymatic activity was determined as described in Methods in the presence of activated D N A with and without pretreatment with RNAase. Only the portion of the activity found to be sensitive to RNAase was used for calculation of the enzyme activity. Fraction

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RNAase-sensitive activity (% of total)

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Characterization of the RNAase-sensitioe DNA polymerase In the presence of the activated calf thymus D N A template, optimal activity of the D N A polymerase is reached with 5 mM Mg 2+ or 1 mM Mn 2+ and 40-100 m M KCI. The enzyme is resistant to 4 0 0 / ~ g / m l phosphonoacetic acid, sensitive to 4 m M N-ethylmaleimide and fully inhibited ( > 98%) by 0.3 m M p-hydroxymercuribenzoate. The optimal p H was found to be in the range of 8.4-9.0. The K m towards dTTP was found to be 2.3.10 - 6 M. A similar K m (3.7.10 6) is found with the high-speed pellet. Addition of r N T P to the reaction mixture did not affect the incorporation of dTMP, however, omitting or replacing one of the dNTPs with r N T P inhibited the incorporation. Activated calf thymus D N A was the only efficient template primer for the reaction. Synthetic templates, as well as RNA, were inefficient templates for the enzyme. The active RNAase-sensitive D N A polymerase was estimated to have a mean molecular weight of 100000, as determined by using four protein markers which were centrifuged in parallel sucrose gradients (Fig. 2B). The presence of RNA molecules in the DNA polymerase complex Since the enzyme sedimented in a well defined peak and was inhibited by RNAase treatment, it was of interest to determine whether this region of the sucrose gradient also contained R N A molecules. Cells in three Roux bottles were labeled with 100 # C i / m l of [32p]orthophosphate for 48 h. The cells were harvested and the enzyme was purified as described in Table II. Polymerase activity, as well as 32p-labeled material cosedimenting with this activity, were completely abolished by RNAase treatment (Fig. 2B). Since RNAase digestion of the 32p-labeled molecules resulted in complete inhibition of the enzymatic activity, it seems that at least some of the R N A molecules which cosediment with the enzyme are required for its activity. It should be noted that efforts to restore activity by addition of various R N A species were unsuccessful.

Comparison of the RNAase-sensitive DNA polymerase to other cellular DNA polymerases by ion exchange chromatography To compare the RNAase-sensitive D N A polymerase activity described here to other known cellular D N A polymerases, RB77 cells were suspended in Buffer A containing Nonidet P-40 (see Methods) and centrifuged for 10 min at 10000 r p m in an SW 50.1 Beckman rotor. The 10000 rpm pellet (nuclear fraction) was dissolved in 2 ml of 0.8 M KCI in 50 mM Tris-HC1, p H 7.8/1 mM E D T A / 1 0 % glycerol/2 mM mercaptoethanol, incubated in ice for 3 h, and centrifuged for 30 min at 45000 rpm in a Beckman 50 Ti rotor. The supernatant was dialyzed overnight against the same buffer without KCi and applied to phosphocellulose or DEAE-cellulose columns. The 10 000 rpm supernatant (cytoplasmic fraction) was further centrifuged at 45 000 rpm, as described in Table II, and the high-speed pellet and the supernatant were treated with ammonium sulfate, dialyzed and subjected to phosphocellulose and DEAE-cellulose chromatography (Fig. 3). The enzyme present in the high-speed pellet, obtained from the cytoplasmic fraction (the RNAase-sensitive D N A polymerase) did not bind to the phosphocellulose and the DEAE-cellulose columns (Fig. 3A and D), and the activities which passed through these columns were completely sensitive to RNAase. However, the enzyme extracted from the nuclear pellet was eluted from the phosphocellulose column by 300 mM KC1 (Fig. 3B) and from the DEAE-cellulose column by 40 mM KC1 (Fig. 3E) and was fully resistant to pretreatment with RNAase. The majority of the enzyme activity present in the 45 000 rpm cytoplasmic fraction supernatant was eluted from phosphocellulose by 150 m M KC1 and did not bind to DEAE-cellulose (Fig. 3C and F). This enzyme was resistant to pretreatment with RNAase. The 45 000 rpm supernatant fraction contained other enzymatic activities, including the D N A polymerase activity described here which did not bind to the columns, and was sensitive to RNAase (Fig. 3C). It can be concluded that the RNAase-sensitive D N A polymerase which is not bound to both DEAE-cellulose and phosphocellulose columns remains RNAase sensitive and it is distinguished from that of the D N A polymerases present in the nuclei or cytoplasm of RB77 cells.

97

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MIACTION Fig. 3. Column chromatography of cellular DNA polymerases on phosphocellulose (A, B, C) and DEAE-cellulose (D, E, F). High-speed pellet extract obtained from the cytoplasmic fraction (A and D), extract obtained from the nuclear pellet (B and E), as well as the 45000 rpm cytoplasmic supernatant (C and F) were chromatographed on phosphocellulose and DEAE-cellulose columns. Aliquots of 5/~! samples from each fraction were used to determine enzymatic activity without (o) or with (O) RNAase pretreatment. The enzymatic reaction was carried out for 30 min, using activated DNA as template; (A) salt concentration.

Discussion

Previous reports from this and other laboratories [12,13,15] have shown the existence of an RNAase-sensitive D N A polymerase in eukaryotic cells. This enzyme was shown to be a DNA-dependent DNA polymerase and not a reverse transcriptase. The template for endogenous activity was shown to be cellular DNA [12]. In the present study, this DNA polymerase was purified 1300-fold and shown to require R N A molecules for its activity even after purification. This RNAase-sensitive D N A polymerase differs in its characteristics from the other cellular DNA polymerases, as well as from the viral reserve transcriptase.

The RNAase sensitivity of the enzyme is retained throughout the purification steps, even when the activity becomes fully dependent on exogenous activated DNA as primer template. It may be speculated that the RNA serves as an initiator molecule for the polymerase activity. This may not be its only role in the reaction, since the enzyme remained fully sensitive to pretreatment with RNAase even when activated DNA was used or introduced during the reaction [12]. Another hypothesis is that the RNA molecule serves to direct the nucleotide triphosphates (or monophosphates) into the correct position on the DNA template [22]. Since RNAase treatment had no effect on the

98

other cellular D N A polymerases, it could be concluded that the effect observed is not due to contamination of the RNAase with proteolytic enzymes. The fact that in the case of 32p-labeled enzyme, acid-insoluble radioactivity and enzyme activity disappeared after RNAase treatment, as well as the lack of activity of inhibited RNAase(bycovin), indicates that the effect observed by RNAase is due to its enzymatic activity on R N A molecules present in the DNA polymerase preparation. It could be suggested that a stable DNA polymerase results from the nonspecific association of RNA molecules with the enzyme fraction. However, it seems unlikely, since purification through DEAE-cellulose or phosphocellulose should be able to remove free nucleic acid impurities. On the other hand, it cannot be ruled out that a specific associated complex between RNA and one of the existing D N A polymerases forms a new type of enzyme somehow involved in DNA replication. N o such association between RNA and cellular D N A polymerases has been reported. Yoshida and Cavalierie [23] have isolated DNA polymerase fl from the nuclear membranes of human lymphoid cells. Although these investigators imphed that the enzyme was RNAase-sensitive, this property was not clearly shown during the different purification steps. In several other studies, RNAase-sensitive enzymes were described, ribonuclease P isolated from E. coli was found to contain an essential RNA component for its activity [24], and some DNA polymerases detected in the cytoplasma of eukaryotic cells fractionated in the presence of detergents were also shown to be RNAase sensitive [10-14]. The activity of this enzyme has been related mainly to transformed cells and to phytohemagglutinin-stimulated lymphocytes [11,13]. However,~ preliminary experiments in our laboratory have shown that a similar enzyme can be found in both transformed cells (e.g., HeLa cells, rat cells transformed by chemical carcinogens, Ehrlich ascites and lymphocytes from patients with; chronic lymphatic leukemia) and nontransformed normal rat cells. The high activity of the RNAase-sensitive D N A polymerase in the cell, the location of the enzyme in membranes, and the association with RNA

molecules may indicate an important function of the RNAase-sensitive DNA polymerase in D N A replication. Further experiments are being carried out in our laboratory in order to gain an understanding of the role of the RNA component in the enzyme activity and its biochemical function.

Acknowledgments We thank Miss. Zohar Ben-Moyal for excellent technical assistance. Part of this investigation was presented in the M. Sc. thesis of Mr. J. Rosenberg. This research was supported by the Ann Langer Cancer Research Foundation and sponsored by the Stiftung Volkswagenwerk.

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