THE JOURNAI, OF BIOI.OGKXI, CHEMISTRY Vol. 239, No. 10, October 1964

Printed in U.S.A.

The Enzymatic Methylation of Ribonucleic Acid and Deoxyribonucleic Acid

IV. THE PROPERTIES OF THE SOLUBLE RIBONUCLEIC ACID-METHYLATING ENZYMES”

JERARD HuRwmz,t ~KARVIN GOLD,$ AND MONIKA ANDERS

From the Department of Microbiology, New Yolk University School of Medicine, New York 16, and the Department of Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, New York 61, New York

(Received for publication, March 27, 1964)

Preceding publications from this laboratory have shown that the methylation of soluble ribonucleic acid is catalyzed by at least six discrete enzymes (l-3) which have been purified from extracts of Escherichia coli W. Two of these enzyme prepara- tions catalyze the formation of l-methylguanine residues in S- RNA,1 while a third produces 7-methylguanine. The others catalyze the methylation of uracil, cytosine, and adenine residues of S-RNA, yielding thymine, 5-methylcytosine, 2-methyladenine, 6-methylaminopurine, and 6-dimethylaminopurine, respectively.

The work presented here describes the properties of these enzymes which catalyze the methylation of different bases of S-RNA. It has been found that each enzyme acts independ- ently, and in all cases, when the reaction comes to a stop with a particular enzyme, further methylation is resumed only after addition of more acceptor S-RNA. The amount of methylated bases present in S-RNA is relatively small. The reactions described here may account for this finding, since there appear to be a limited number of methylation sites for a given enzyme and S-RNA.

EXPERIMENTAL PROCEDURE

The purification and assay of the methylation enzymes have been described in the preceding paper (3). The following com- pounds were commercial preparations: polyriboadenylate and polyribocytidylate (Miles Chemical Company, Clifton, New Jersey); rat liver S-RNA (General Biochemical Corporation, Chagrin Falls, Ohio) ; 14Cformaldehyde (Volk Radiochemical Company, Chicago); DNase, RNase, spleen phosphodiesterase, and calf thymus DNA (Worthington Biochemical Corporation, Freehold, New Jersey) ; and %-methyl-labeled L-methionine (Tracerlab, Inc., Waltham, Massachusetts). The following com- pounds were obtained as gifts: tetrahydrofolic acid (Dr. M. J. Osborn of this Department) and S-adenosyl-L-homocysteine (Dr. J. A. Duerre of the Argonne National Laboratory, Argonne, Illinois). The latter compound, as indicated by Dr. Duerre, was

* This research was supported by grants from the National In- stitutes of Health, the National Research Council, and the Public Health Research Council of the City of New York, Inc.

t Senior Postdoctoral Fellow of the National Institutes of Health; American Cancer Society Professor of Molecular Biology, July 1963.

$ Fellow of the Jane Coffin Childs Memorial Fund for Medical Research, 1962 to 1963.

1 The abbreviation used is: S-RNA, soluble or transfer ribo- nucleic acid.

contaminated with a small amount of adenosine (approximately 2%). No correction was made for this contamination, and the amount of S-adenosyl-L-homocysteine was estimated spec- trophotometrically with the use of the extinction coefficient reported for adenosine. The labeled compounds 5, B-dimethyl- benzimidazolylcobamide 14C-methyl (14C-methyl-B12) and Nj- 14C-methyltetrahydrofolic acid (14C-5-CHs-folate-H4) were ob- tained as gifts from Dr. Herbert Weissbach of the National Institutes of Health. Venom phosphodiest,erase was purified according to Koerner and Sinsheimer (4) from crude Crotalus adamanteus venom purchased from Ross Allen’s Reptile In- stitute, Silver Springs, Florida. One unit of phosphodiesterase activity is defined as the amount that releases 1 pmole of mono- nucleotide per hour from a pancreatic DNase digest of thymus DNA.

S-RNA, deficient in methyl groups, from E. coli K12-5%161- F-, methionine-, was prepared as previously described (3). Ribosomal RNA from this same organism, grown with either excess or limiting methionine, was isolated from pellets obtained by high speed centrifugation of crude extracts and purified as follows. The pellets were resuspended in 0.01 M Tris buffer, pH 8.0, containing 0.01 M MgClz and 5 pg of DNase per ml, and were centrifuged at 30,000 x g for 15 minutes. The supernatant solution was then recentrifuged at 78,000 x g for 3 hours, and the cycles of low and high speed centrifugation were repeated. The final pellet was resuspended in 0.01 M Tris buffer, pH 8.0, mixed with an equal volume of 80% phenol, and shaken vigor- ously for 30 minutes at 38”; the aqueous phase was removed and treated twice more with phenol as above. The final aqueous phase was then extracted three times with ether, adjusted to 2% with potassium acetate buffer, pH 5.1, and mixed with 2 volumes of ethanol. After 30 minutes at O”, the flocculent precipitate was collected, dissolved in 0.01 M Tris buffer, pH 8.0, and dialyzed overnight against the same buffer. This procedure, when ap- plied to ribosomes from cells grown in a methionine-deficient medium, yielded RNA which was methylated in vitro with the purified S-RNA methylation enzymes to a relatively low extent (approximately 5% of that observed with comparable amounts of S-RNA). This methyl-accepting activity was separated from the ribosomal RNA by chromatography on Sephadex G-200 (Pharmacia, Uppsala). The low level of methylation of the ribosomal RNA preparations appeared to be due to contamina- tion with S-RNA. Chromatography on Sephadex columns was carried out as follows. Ribosomal RNA, 586 optical density

3474

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October 19G4 J. Hurwitz, M. Gold, and Al. Anders 3475

units as measured at 260 mp, was added to a column (28 x 3 cm) of Sephadex G-200 suspended in 0.05 M ammonium acetate buffer, pH 5.0, and then eluted with the same solvent. Fractions of 3 ml were collected, and two discrete peaks of ultraviolet- absorbing material were isolated. RNA of the first peak (21 ml), accounting for 485 optical density units, was precipitated by the addition of 0.5 ml of 20% potassium acetate buffer, pH 5.1, and 40 ml of ethanol. The precipitate was collected, dissolved in 0.02 M Tris buffer, pH 8.0, and dialyzed overnight against 200 ml of the same buffer solution. After dialysis, 465 absorbance units were recovered. The material of the second peak from the Sephadex column was active as a methyl group acceptor in the assay as described above, and thus was presumed to contain S-RNA.

RNA of tobacco mosaic virus was isolated from the virus (kindly supplied by Dr. C. A. Knight of the University of Cali- fornia, Berkeley) by phenol treatment (5). RNA of turnip yellow mosaic virus was a gift of Dr. J. Ofengand (Department of Biochemistry, University of California School of Medicine), and RNA isolated from bacteriophage f2 was supplied by Dr. J. T. August of this Department. Synthetic polyuridylate-guanylate mixed polymers were a gift from Dr. L. A. Heppel of the National Institutes of Health. Methyl-deficient DNA was prepared as described by Marmur (6) from the relaxed strain grown under conditions of methionine limitation. Liquid scintillation count- ing was carried out as previously described (7). No correction has been applied to any of the results for loss of activity due to quenching.

RESULTS

Properties of PuriJied Enzymes

Eflect of pH on Rate of Reaction-Optimal activities for the different methylating enzymes were found in the following pH ranges: guanine I, 8.0 to 8.5 in both Tris and triethanolamine buffers; guanine II and cytosine, triethanolamine buffer, pH 8.0 to 9.0; adenine and uracil, t,riethanolamine buffer, pH 8.5 to 9.0; and guanine III, dimethylglutarate buffer, pH 7.5 to 8.0.

Requirement for Metals and Sulfhydryl Agents-All enzymes showed some activity in the absence of Mg++ (Table I). The methylation of cytosine was not affected by Mg++, while the uracil-met,hylating and the guanine-methylating activities I and II were increased approximately 4-fold, and guanine-methylating enzyme III 2-fold, by the presence of Mg++. Methylation of adenine was stimulated only 2-fold by Mg++. Similar effects were observed with Ca++ or Mn++ in place of Mg+f although there were quantitive differences; Ni++ and Zn++ were inhibitory. In general, spermine did not stimulat,e, except for a slight effect with the uracil-methylating enzyme.

The enzyme preparations were routinely stored in the presence of 2-mercaptoethanol; in some cases, there was a marked decrease in methylation activity when this compound was omitted from the reaction mixtures, especially in t,he case of the uracil-methyl- ating enzyme. The addition of p-hydroxymercuribenzoate decreased the activity of all the enzyme fractions tested.

Eject of Concentration of S-Adenosylmethionine and X-RNA- The apparent affinities of the substrates (8) varied among the different enzyme fractions. These values (as ribonucleotide residues x 10P4 M) for methyl-deficient S-RNA of E. coli were: guanine I, 8.3; guanine II, 6.7; uracil, 2.9; cytosine, 5.0; guanine III, 2.1; and adenine, 16 (Fig. ld). The latter enzyme fraction

Reaction mixture -

(

Effect of metals No metal.. Mg++ Ni++ Zn++ Ca++ Mn++ Mg++ + spermine.. Spermine (no metal: 1.

Effect of sulfhydry 1 agents

TABLE I

Effect oj metals and suljhydryl agents Reaction mixtures (0.25 ml) contained 10 mMmoles of IaC-

methyl-labeled S-adenosylmethionine (2.2 X lo7 c.p.m. per pmole); 10 Mmoles of triethanolamine buffer, pH 8.0 (guanine I, guanine II, cytosine, and guanine III enzymes) or pH 8.8 (uracil- and adenine-methylating fractions) ; 1 pmole of metal salt, as in- dicated; 2 pmoles of 2-mercaptoethanol; 410 mpmoles of nucleo- tide residues as methyl-deficient E. coli S-RNA; and, where indicated, 1 pmole of spermine, 1 pmole of p-hydroxymercuri- benzoate, and the following amounts of methylating enzyme preparation (in micrograms of protein) : guanine I, 0.61; guanine II, 0.56; uracil, 2.3; cytosine, 0.69; adenine, 0.73; and guanine III, 0.35. Reaction mixtures were incubated for 15 minutes at 38”, and the amount of acid-insoluble radioactivity was measured as described previously. Reaction mixtures contained 1 Mmole of MgCl2 in the three cases in which the effects of sulfhydryl agents were examined.

11 6 4G 28 12 7 3 <l

34 32 53 29

9 11 2 2

74 18 69 36 I1 8

5 3 73 34 58 22 41 24 51 18

150 320 -*

356 42

111 266

Complete system. Omit 2-mercapto-

ethanol. Omit 2-mercapto-

ethanol + p-hy- droxymercuriben- zoate

42 32

34 25

45 175

5 <1 183 84

111 85

173

12

9

71 38 328

76 20 120

9 i

<1 11 5 -

* A dash indicates that the effects of such additions were not studied.

shows the lowest affinity for S-RNA, while guanine-methylating enzyme III shows the greatest. The affinity constants (X lo+ M) for X-adenosylmethioninc were the following: guanine I, 2.4; guanine II, 6.7; uracil, 1.8; cytosine, 1.3; adenine, 1.1; and guanine III, 1.6 (Fig. 1B).

Requirements of Reaction--The addition of RNase (0.5 pg) abolished methylation of S-RNA, while DNase (0.5 pg) was without effect. In the reaction mixtures employed, S-adenosyl- methionine could not be replaced by methionine and ATI’ (9), in contrast to crude extracts, in which methionine and ATP were effective. Other methyl donors which have also been found to be incapable of replacing X-adenosylmethionine were 14C-formalde- hyde plus tetrahydrofolic acid, 14C-methyl B12, and r4C-5-CHa- folate-Hr. These methyl donors were tested both with crude extract,s of E. coli and with the partially purified enzyme frac-

tions. All other nucleic acid derivatives, with the exception of a

variety of S-RNA preparations (3), were inactive with the puri- fied enzymes. This list includes methyl-deficient DNA of E.

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3476 Enzymatic Methylation of RNA and DNA. IV Vol. 239, No. 10

-4.0 -30 -20 -1.0 1.0 2.0 3.0 40 60 VS

-1.0 -015 -0.50 -0.25 0.25 050 0.75 10 I.25 1.50 I/S X10 IS=mpmolcr per ml)

FIG. 1. Affinity constant for the substrates of the reaction. Reaction mixtures were as described in Table I. The abbreviation S refers to micromoles of nucleotide residues as methyl-deficient S-RNA. When the affinity constant for S-RNA was measured (A), 10 mpmoles of S-adenosylmethionine were added. When the constant for S-adenosylmethionine was measured (B), 200 mpmoles of nucleotide residues as methyl-deficient E. coli S-RNA were added for all enzymes except the adenine-methylating fraction and guanine-methylating enzyme III; for the latter two, 436 and 410 mpmoles of nucleotide residues as S-RNA were added, respec- tively.

coli K12-58-161-F-, methionine-, which indicates that, in all

cases, the purification procedure resulted either in the inactiva- tion or in the removal of the DNA methylation activity (1). A variet,y of synthetic as well as natural RNA polymers were tested

wit,h the ammonium sulfate fraction (prior to phosphocellulose

chromatography). These included polyadenylic acid, poly- cytidylic acid, polyuridylic acid, polyinosinic acid, copolymers of

adenylic acid and uridylic acid (alternating) and of guanylic acid and cytidylic acid (both made in the RNA polymerase reaction

in the presence of the corresponding DNA primers), tobacco mosaic viral R?JA, turnip yellow mosaic viral RNA, and RNA from bacteriophage f2. All were inactive as methyl group ac-

ceptors. ?jone of the purified enzyme fractions catalyzed the methylat,ion of the 5’-mononucleotides CMP, AMP, UMP, or

GMP. Ribosomal RNA isolated from normal or methyl-deficient

cells was also inactive with the purified enzyme fractions.2 Simi- lar observations regarding the inactivity of ribosomal RNA have been reported by Svensson et al. (10) and Fleissner and Borek

(11). Stoichiometry of Reaction-The stoichiometry of the reaction

was studied by measuring the amount of S-adenosylmethionine remaining after the reaction catalyzed by the uracil-methylating enzyme and the cytosine-methylating enzyme (Table II). Close correspondence was observed between the amount of iGmethy1 group incorporated into RNA and the decrease in X-adenosyl-

methionine. The other reaction product is presumed to be S- adenosylhomocytosine, which, as described below, is an inhibitor of the reaction.

2 Enzymes capable of catalyzing the methylation of ribosomal RNA have been isolated from E. coli. These systems will be de- scribed at a later time.

Extent of Methylation of X-RNA with Different Enzyme Frac- tions-Reaction rates and yields with the purified enzyme frac- tions were measured with methyl-deficient S-RNA. The initial rate of methylation was proportional to the amount of enzyme added. In all cases, when large amounts of enzyme were added or when incubation was prolonged, finite amounts of methyl group were incorporated (Fig. 2). The extent of methylation was directly proportional to the amount of S-RNA added. The extent of methylation of S-RNA by the different enzyme frac- tions varied; thus, the yield of incorporation was nearly 10 times higher with the uracil enzyme than with most of the other enzyme

fractions. The incorporation of methyl group catalyzed by the adenine-methylating fraction was nearly 35 times lower than that found in the case of the uracil-methylating enzyme.

The following experiment suggested that the reaction comes to a halt because the sites which can be methylated are exhausted during t,he reaction. With each enzyme fraction, two identical reaction mixtures were used. After the reaction had ceased,

more enzyme was added to one reaction mixture while additional methyl-deficient S-RNA was added to the second. Only in the latter case was there further incorporation of labeled methyl groups. These results are summarized in Fig. 3. In most cases, the rate of reaction during the second response to acceptor S-RNA addition was less than that observed during the initial phase of the experiment. Control mixtures in which enzyme and S-RNA

were incubated in the absence of S-adenosylmethionine did not indicate inactivation of the enzyme. The decreased rate of methylation observed in the second phase of the experiment

(Fig. 3) is most pronounced in the case of the uracil-methylating

TABLE II

Stoichiometry oj methylation reaction

Reaction mixtures (0.25 ml) contained 0.97 mpmole of S- adenosylmethionine (2.28 X lo7 c.p.m. per pmole); 1 pmole of MgC12; 10 pmoles of triethanolamine buffer, pH 8.8; and 2 Mmoles of 2.mercaptoethanol; where indicated, 175 mpmoles of nucleotide residues as methyl-deficient E. coli S-RNA and 0.16 unit of uracil- methylating enzyme, or 0.34 unit of cytosine-methylating enzyme and 375 mMmoles of methyl deficient S-RNA nucleotide residues, were added. The reaction mixtures were incubated for 300 minutes, after which 0.05 ml of 1 N HClOd and 0.02 ml of 0.3yn albumin were added, the suspensions were centrifuged, and the supernatant solutions were decanted. The pellets were washed once more with 0.1 ml of 7% HClOd, and the supernatant solutions and washing were combined. To the combined solutions, 5 pmoles of unlabeled S-adenosylmethionine were added, and 0.05 ml of this solution was subjected to electrophoresis on paper at pH 3.5 in 0.05 M ammonium formate buffer for 1 hour. The ultra- violet-absorbing material which migrated toward the cathode was cut out and counted directly in a scintillation solution. Con- trols in which S-RNA was omitted from the reaction mixture were treated in exactly the same way.

Reaction mixture

Uracil-methylating enzyme Omit S-RNA.. Add S-RNA.

Cytosine-methylating enzyme Omit S-RNA. Add S-RNA.

‘GMethyl group

incorporated

<O.OOl 0.97 0.38 0.55

<O.OOl 0.92 0.22 0.65

0.42

0.27

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October 1964 J. Hurwitx, M. Gold, and M. Anders 3477

FIG. 2. The extent of methylation of S-RNA with different en- zyme fractions. The reaction mixtures (0.5 ml) were as follows. Guanine-methylating enzyme I : 30 mpmoles of S-adenosylmethi- onine (1.7 X 107 c.p.m. per rmole), 3 pmoles of MgC12, 6 pmoles of 2-mercaptoethanol, 30 pmoles of triethanolamine buffer, pH 8.0, and 0.08 unit of enzyme. The curves represented by the open and closed circles contained 1.02 and 0.204 pmoles, respectively, of nucleotide residues as methyl-deficient E. coli S-RNA. The reac- tion was initiated by the addition of enzyme and, as indicated, 0.05-ml aliquots were removed, acidified with 5976 trichloroacetic acid, and passed through Millipore filters; the Millipore filters, containing S-RNA, were further washed with 1% trichloroacetic acid prior to drying and counting. Guanine-methylating enzyme II : the conditions were as described for guanine-methylating en- zyme I, except that 1.02 and 0.510 pmoles of nucleotide residues as S-RNA were added, with 0.11 unit of enzyme. Uracil-methylating

enzyme. This appears to be related t.o the inhibition of methyla- tion by S-adenosylhomocysteine, which is presumed to be formed as one of the products of the reaction.

Activity of Enzymes on S-RNA Pretreated under Different Conditions-The purified enzymes were tested with S-RNA which had been treated with diesterases, acid, and heat (Table III). The rate and yield of the reaction were most influenced by pretreatment of S-RNA with spleen phosphodiesterase. While it was suspected that this enzyme preparation may have cont.ained small amounts of endonuclease, the enzymatic reac- tion by which guanine is converted to 1-methylguanine in S-RNA appears to be affected most by this pretreatment, while there is only a 50% reduction of both the rate and yield of methylation of S-RNA catalyzed by the other enzyme fractions. Venom phosphodiesterase treatment, heating, or acid precipitation of the RNA had little effect on its substrate activity.

Degradation of 14C-Methyl-labeled S-RNA with Snake Venom Phosphodiesterase-From the results presented in Table III, it would appear that extensive removal of the terminal ribonucleo- tides by venom phosphodiesterase hardly affects the rate or extent of methylation. This suggests that methylation does not occur exclusively at the ends of S-RNA chains. This conclusion was supported by measurement of the rate of formation of both acid-soluble, methyl-labeled nucleotides and acid-soluble, ultra- violet-absorbing material during venom phosphodiesterase treat- mer t (Fig. 4). In all cases, there was correspondence between

enzyme: the conditions were the same as in the case of guanine- methylating enzyme I, except that 30 pmoles of triethanolamine buffer, pH 8.8, and 0.67 unit of uracil-methylating enzyme were added. Cytosine-methylating enzyme: the reaction mixture was as described for guanine-methylating enzyme I, except that MgClz was omitted and 0.10 unit of enzyme was used. Adenine- methylating fraction: the volume of the reaction mixture was in- creased to 0.75 ml; 30 mpmoles of S-adenosylmethionine (2.28 x 107

c.p.m. per @mole), 6 pmoles of MgC12, 60 pmoles of triethanolamine buffer, pH 8.8, and 0.08 unit of enzyme were used; and 4.86 and 1.62 pmoles of nucleotide residues as S-RNA were added as ac- ceptor. Guanine-methylating enzyme III: the reaction mixture was as described for guanine-methylating enzyme I with 0.37 unit of guanine-methylating enzyme III and 1.025 and 0.41 rmoles of nucleotide residues as S-RNA.

the rates of release of radioactivity and ultraviolet-absorbing material, indicating that methylation of S-RNA does not occur preferentially at either the 3’-hydroxyl or the 5’-phosphate end of the S-RNA molecule. These results also suggest that the distribution of labeled methyl groups is more or less random in the heterogenous population of S-RNA molecules.

Extent of Methylation of S-RNA by Combination of Enzymes- The combined action of the methylation enzymes, in a single reaction mixture, resulted in t.he formation of S-RNA containing W-labeled methyl derivatives of all the bases.3 This is in keeping with the observation that each of the enzymes acts independently. The result of combined action of the methylat- ing enzymes has been studied quantitatively (Table IV). It is evident that when two of the methylation enzymes are combined, there is an additive yield of methylation, except in the case of guanine-methylating enzymes I and II. In this case, common sites appear to be methylated by t.hese two different enzyme fractions, and they are not additive. An experiment to elucidate this point consisted of the following steps.

Step 1: K-S-Adenosylmethionine

+ S-RNA guanlne ‘> 12C-methyl-S-RNA enzyme

3 Unpublished experiments of the authors.

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3478 Enzymatic Methylation of RNA and DNA. IV Vol. 239, No. 10

iBrlJRACIL

1.5

12

7s

B 0.9

: 0.6

0.3

0- 30 60 SO 120 150 180 210

FIG. 3. Extent Of

30 60 SO 120 150 180 210

0.35rGUANINE I

30 60 SO 120 150 180 TIME (MINUTES)

030 GUANINE II

025

0.20

015

010

005

’ 30 60 SO 120 150 180 TIME (MINUTES)

the methylation and the effect of addition of the mixtures contained 40 rmoles of triethanolamine buffer, pH 8.8,0.44 unit of enzyme, and 224 mwmoles of nucleotide residues as methyl-deficient S-“RNA. At 90 minutes, 306 mpmoles of nucleo- tide residues as S-RNA and 0.33 unit of enzyme were added. Cytosine-methylating enzyme: 0.13 unit of enzyme was added while all other conditions were as described for guanine-methylat- ing enzyme I. At 90 minutes, 0.10 unit of enzyme and 306 mpmoles of nucleotides residues were added. Adenine-methylating frac- tion: 40 rmoles of triethanolamine buffer, pH 8.8, 1.2 @moles of nucleotide residues as S-RNA, and 0.08 unit of enzyme were added. After 120 minutes, 0.9 rmole of nucleotide residues and 0.08 unit of enzyme were added. All other conditions were as described for guanine-methylating enzyme I. Guanine-methylating enzyme III: the mixture contained 40 pmoles of triethanolamine buffer, pH 8.0,820 mpmoles of nucleotide residues as S-RNA, and 0.8 unit of enzyme; all other additions were made as above. At 120 min- utes, 820 mflmoles of nucleotide residues and 0.8 unit of enzyme were added.

enzyme or RNA. Unless otherwise indicated, reaction mixtures (1.0 ml) contained 40 mpmoles of S-adenosylmethionine (2.6 X 107 c.p.m. per Fmole), 408 mpmoles of nucleotide residues as methyl- deficient E. coli S-RNA, 4 pmoles of 2-mercaptoethanol, 4 pmoles of MgCls, enzyme, and buffer. Two reaction mixtures were pre- pared containing the above ingredients; at the time indicated by the arrows, additional enzyme was added to one and S-RNA to the other. Reactions were-followed by removal of O.l-ml aliquots, which were nrecinitated with acid (HClO,) and washed bv re- peated centrifugation as previously described. Guanine-methyl- ating enzyme I: 40 mpmoles of the triethanolamine buffer, pH 8.0, and 0.12 unit of enzyme were added. At 90 minutes, 0.10 unit of guanine-methylating enzyme I and 306 mbmoles of ribonucleotide residues as S-RNA were added. Guanine-methylating enzyme II: additions were made as in the case of guanine-methylating enzyme I, except that 0.09 unit of enzyme was added at the indi- cated time to one reaction mixture. Reaction mixtures were treated as previously described. Uracil-methylating enzyme:

The nonradioactive S-RNA was isolated after acid precipitation bacteria suggest that they do not share all of the same methyla-

and dialysis. tion sites (2).

Step 2: nC-Methyl-S-adenosylmethionine

+ 12C-methyl-S-RNA w guanine I1 14C-methyl-S-RNA enzyme

When these procedures were carried out, only 10% of the expected methylation occurred in Step 2 when compared with

S-RNA treated with guanine-methylating enzyme I in the ab- sence of S-adenosylmethionine. There was no detect~able

methylation when guanine-methylating enzyme I was substituted

for guanine-methylating enzyme II in Step 2. When the enzyme treatment was reversed so that Step 1 was

carried out with guanine-methylating enzyme II, 14y0 of the expected methylation occurred in t,he second step with guanine-

methylating enzyme I. However, in this case, a control in which guanine-methylating enzyme II was added in the second

reaction in place of guanine-methylating enzyme I resulted in

8% of the expected extent of methylation. It appears that the

guanine enzymes may have common sites for methylation, although previous results with S-RNA preparations from different

Attempts at Reversal of Reaction and Inhibition by S-Adenosyl- homocysteine-Reversal of the methylation reaction could not be demonstrated. The addition of S-adenosylhomocysteine after exhaustive methylation catalyzed by the uracil enzyme did not

produce a decrease in the amount of acid-insoluble radioactivity (Fig. 5). However, when S-adenosylhomocysteine was initially included in the reaction mixture, the rate of methylation of S- RNA was markedly depressed. The degree of inhibition of methylation catalyzed by the different enzymes varies con- siderably (Fig. 6). Fifty per cent inhibition of the reaction occurred at the following concentrations of S-adenosylhomo- cysteine (X 10P5 M): uracil, 0.4; cytosine, 0.7; guanine III, 0.85; adenine, 3.0; guanine II, 4.3; and guanine I, approximately 16. The effect of S-adenosylhomocysteine was relatively specific, and replacement of this compound with much higher levels of adenosine or L-homocysteine did not appreciably affect the reac- tion (Table V).

The inhibition of methylation by X-adenosylhomocysteine appears to be competitive (Fig. 7) and is reversed by increasing concentrations of S-adenosylmethionine. With the uracil

4.0 GUAN INE III

30

0.30 ADENINE FRACTION

0.25

0.20

0.15

0.10

0.05

’

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October 1964 3479 J. Hurwitx, Al. Gold, and M. Anders

TABLE III

E$ect of various agents on S-RNA and its acceptor ability

Rates and yields of methylation were measured in the routine triethanolamine buffer, pH 8.0. This solution, which is listed assay mixture with 415 mpmoles of ribonucleotide residues as below as the venom diesterase-pretreated RNA, contained 38 S-RNA for all enzymes, except that 206 mpmoles of ribonucleotide optical density units (measured at 260 mp). Spleen phosphodies- residues were added with the uracil-methylating enzyme. These terase: reaction mixtures (0.3 ml) contained 25pmoles of potassium amounts of ribonucleotide residues were added in all cases, even phosphate buffer, pH 6.5, 50 optical density units (measured at when the pretreatment of S-RNA resulted in loss of ultraviolet- 260 rnh) of S-RNA, and 0.36 units of spleen phosphodiesterase. absorbing material. Pretreatment was done as follows. Venom All other conditions were as described above. The acid-soluble, diestcrase: reaction mixtures (0.3 ml) containing 50 optical density ultraviolet-absorbing material produced after incubation with units (at 2GO rnp) of S-RNA, 25 pmoles of triethanolamine buffer, spleen phosphodiesterase accounted for 11.6 optical density units, pH 8.5, and 10 units of purified snake venom phosphodiesterase while 34.4 optical density units were recovered in the acid-insolu- were incubated for 30 minutes. The reaction mixture was then ble material. RNA heated to 100”: the RNA solution (50 optical acidified with 0.05 ml of 70/c HClOh and centrifuged. The super- density units at 260 rnp) in 0.025 M Tris buffer, pH 7.5, was heated natant solution contained a total of 6.4 optical density units (at at 100” for 5 minutes and rapidly cooled. No detectable acid- 260 mp). The acid-insoluble material was washed once with 1% soluble, ultraviolet-absorbing material was formed by this treat- HC104 and, after centrifugation, was dissolved in I ml of 0.05 M ment.

Pretreatment of S-RNA

None ....................................... Venom diesterase ............................ Spleen phosphodiesterase .................... Heating to 100”. ............................. Acid precipitation ...........................

Guanine I-methylat- Guanine II-methylat- Uracil-methylating ing enzyme ing enzyme ~~Z~~~

/ Yield 1 Rate 1 Yield 1Rafe Rate Yield

176 0.204 151 0.220 382 0.900

133 0.197 109 0.176 273 0.640 8 0.013 13 0.042 203 0.452

131 0.202 134 0.231 250 0.800 103 0.236 108 0.171 326 0.834

C”-CH -S-RNA formed with C’4-CH,-S-RNA formed with ,oo G”ANI:E I ENZYME 6 ,yRACIL ENZYME p.

SO c

90 150 210 270 330 390 ’ 60 120 180 240 co -rl~E (MINUTES)

C1’%ti3-+~~~ formed with 2 ,OO-GUANINE II ENZYME

z

’ 30 90 150 210 270 330 390

mpmole

Cytosine-methylat- Adenine-methylat- ing enzyme ing fraction

Rate

QCWkS,

wlhr

232 220 106 220 238

Yield

I I

Rate Yield _____

0.214 86 0.046 0.210 61 0.048 0.123 34 0.024 0.242 69 0.046 0.268 65 0.052

64 120 180 240 ” 60 120 180 240 -rl~E (MINUTES)

FIG. 4. Degradation of 14C-methyl-labeled S-RNA with venom and acid-soluble, ultraviolet-absorbing material produced. COOL- phosphodiesterase. S-RNA was completely saturated with W- trols were simultaneously run in which venom phosphodiesterase methyl groups in the presence of excess enzyme. In most cases, was omitted from one tube and S-RNA omitted from another. reaction mixtures (0.5 ml) for degradation of methyl-labeled S- Incubation mixtures in which changes were made were the follow- RNA contained 0.49 pmole of nucleotide residues as S-RNA with ing: thymine-containing product, the additions were the same as 10,000 c.p.m., 25 pmoles of triethanolamine buffer, pH 8.8, and 10 those above, except that the S-RNA added contained 82,500 c.p.m.; units of venom phosphodiesterase. Aliquots (0.05 ml) were re- adenine fraction product, the conditions for degradation were the moved at indicated times and treated with 0.1 ml of 7$& HClO,, 0.6 same, except that the reaction mixture volume and aliquots re- ml of HgO, and 0.2 ml of salmon sperm DNA containing 0.19 pmole moved were increased a-fold. The amount of radioactivity added of deoxyribonucleotide residue. After 5 minutes at O”, reaction in the form of labeled S-RNA was 5,200 c.p.m. The above degrada- mixtures were centrifuged; aliquots of the supernatant solution tion was not carried out with the product obtained with guanine- were used for determining the amount of acid-soluble radioactivity methylating enzyme III.

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3480 Enzymatic Methylation of RNA and DNA. IV Vol. 239, No. 10

TABLE IV

Extent of methylation of S-RNA by combination of methylating enzymes

The reaction mixtures (0.5 ml) contained 20 pmoles of tri- ethanolamine buffer, pH 8.5, 4 pmoles of 2-mercaptoethanol, 20 mpmoles of 14C-S-adenosylmethionine (2.2 X 1Or c.p.m. perrmole), 4 pmoles of MgClz, 217 mpmoles of ribonucleotides as methyl- deficient S-RNA, and the following amounts of enzyme (in units) : guanine I, 0.04; guanine II, 0.07; uracil, 0.67; cytosine, 0.10; and adenine, 0.07. In Experiment 3, 513 mpmoles of S-RNA nucleo- tide residues and 0.35 unit of guanine-methylating enzyme III were added. The reactions were carried out for 3 hours at 38”. The extent of incorporation was measured as previously described.

Methylating enzyme(s) added

Experiment 1 Uracil................................. Guanine I. ............................ Guanine I + uracil ...................

Change due to uracil. Adenine. ............................. Adenine + Guanine I. .................

Change due to guanine I. ............ Experiment 2

Uracil................................. Cytosine ............................. Uracil + cytosine .....................

Change due to uracil ............... Guanine II. ........................... Guanine II + cytosine .................

Change due to cytosine. ............. Experiment 3

Guanine I ............................. Guanine II ......... ................. Guanine III ........................... Guanine I + guanine III. ............. Guanine II + guanine III. ............

Experiment 4 Guanine I ............................. Guanine II. ........................... Guanine I + guanine II. ..............

14C incorporation

0.89

0.086 0.96

0.87 0.029

0.103 0.074

0.86 0.10 0.99 0.89 0.079 0.187 0.108

0.21 0.24 1.17 1.37 1.42

0.080 0.077 0.087

enzyme, the K; for S-adenosylhomocysteine from Lineweaver-

Burk (8) plots (Fig. 7) is 2 X 10-s M.

DISCUSSION

The methylation reactions catalyzed by the enzymes described in this paper are specific with respect to both the methyl group acceptor, i.e. S-RNA, and the methyl group donor, i.e. S-ad- enosylmethionine. While S-RNA preparations from a variety

of sources act as acceptors, no other RNA species have been found to be active. With this high degree of specificity, these enzymes can be useful for detecting the presence of S-RNA in

mixtures of various species of RNA. We have used this test to demonstrate, with several DNA primers, that a small but signifi- cant amount of RNA with this property is formed in the RNA polymerase reaction (9, 12).

From the data presented, it is seen that there are limited sites available for methylation in S-RNA. The extent of methyla- tion of methyl-deficient S-RNA catalyzed by the enzymes was the following (in percentage of nucleotide residues methylated) : guanine I, 0.04; guanine II, 0.034; uracil, 0.50; cytosine, 0.05;

adenine, 0.013; and guanine III, 0.19. When these sites are saturated, no further methylation occurs even when additional enzyme is added. However, when additional S-RNA is added. there is a resumption of methylation.

The exact requirement for methylation is presently unknown. The enzymes catalyze extensive methylation independent of one another, except, for guanine-methylating enzymes I and II, which appear to have similar requirements since the extent of methylation by these two enzymes is far from additive. The fact that these two enzymes both produce 1-methylguanine residues is also distinctive. The presence of two enzymes which lead to the formation of 1-methylguanine residues at presumably the same site in the S-RNA is of interest and requires further study.

S-ADENOSYL HOMOCYSTEINE ADDED

30 60 90 120 150 160

TIME (min.)

FIG. 5. Attempts at reversal of reaction. The reaction mixture (1.0 ml) contained 30 mpmoles of S-adenosylmethionine (1 X 1Or c.p.m. per pmole), 184 mpmoles of ribonucleotide residues as methyl-deficient E. coli S-RNA, 2 pmoles of MgC12, 2 pmoles of 2-mercaptoethanol, 40 pmoles of triethanolamine buffer, pH 8.8, and 0.17 unit of uracil-methylating enzyme. At 90 minutes, 34 mpmoles of S-adenosylhomocysteine were added (Curve A); in Curve B, the same amount of S-adenosylhomocysteine was added prior to enzyme addition, As indicated, 0.05-ml aliquots were removed from reaction mixtures of A, and O.l-ml aliquots were removed from those of B; the amount of acid-insoluble radioac- tivity was then measured, as previously described.

URACIL

J--kk+F 10

S-ADENOSYL HOMOCYSTEINE 0.4~16~)

FIG. 6. Inhibition of enzyme reactions by S-adenosylhomocys- teine. Reaction mixtures were as described in Fig. 5 with the amount of S-adenosylhomocysteine as indicated. The incorpora- tion of methyl groups after 20 minutes of incubation (in micro- micromoles) in the absence of S-adenosylhomocysteine was the following: guanine I, 39; guanine II, 62; adenine, 33; cytosine, 120; uracil, 116; and guanine III, 330.

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October 1964 J. Hurwitx, ill. Gold, and Al. Anders 3481

The ability of the methylation enzymes to detect and methyl- ate S-RNA within a mixture of differing species of RNA suggests that these enzymes have a high affinity for S-RNA. It would seem likely, a priori, that this specificity would be governed by the secondary structure of the RNA. These properties were apparently not altered by the diesterase treatments described in Table III, despite the fact that approximately 15% of the nucleo- tides were released by the venom as well as by the spleen phos- phodiesterase treatment. These observations suggest that the oligonucleotide residues at the 3’-hydroxyl end of the S-RNA molecule may not be important. In agreement with these results, Svensson et al. (10) have demonstrated that S-RNA oxidized with periodate is fully capable of accepting methyl groups. It has been established by others that acid precipitation as well as heat,ing to 100” have virtually no permanent effect on the secondary structure of S-RNA (13).

li‘rom dat.a on the extent of enzymatic methylation, one would expect that the most common methylated base in S-RNA would be thymine. This observation is in keeping with the report by Dunn, Smith, and Spahr (14). On the other hand, our data predict that methylated adenine derivatives would be presen

TABLE V

Specijkity oj S-adenosylhomocysteine inhibition of methylation

Reaction mixtures (0.25 ml) contained 10 mpmoles of S-ade- nosylmethionine (2.28 X lo1 c.p.m. per pmole); 500 mpmoles of nucleotide residues as S-RNA; 1 pmole of MgClz; 2 pmoles of 2-mercaptoethanol; 10 pmoles of triethanolamine buffer, either pH 8.0 or 8.8; and L-homocysteine, adenosine, or S-adenosylhomo- cysteine as indicated. Reaction mixtures were incubated at 38” for 15 minutes; all other conditions were as previously described.

Enzyme fraction

Guanine I. Guanine II. Uracil Cytosine.. Adenine Guanine III.

I

-

Incorpo- ration

with no nhibitor

mpmoles

202 135 130 120

33 330

I-Homocysteine, 2 x 10’ M

Incorpo- Inhibi- ration tion

~~

mpmoles %

187 8 130 <5 132 0 130 0

30 10 310 6

- I

Inhibitor added

Adenosine, 2 x 10-a M

- I S-Adenosyk-

homocysteine, 1.25 X 10-d 3d

Incorpo ration

Inhibi- tion

Incorpo- Inhi- ration bition

WQ.l??&OlC-$ % ~~P%Ol~S % 173 12 170 16 96 26 65 52

112 14 7 95 132 0 9 92 30 10 13 57

360 0 130 61

200

160

t

I/V 120

80 S-adenosylhomocysieine

I 1 I I I I

20 40 60 80 100 i20 I/S (umoles of S-odenosylmethionine)

FIG. 7. Competitive inhibition by S-adenosylhomocysteine. Reaction mixtures were as described in Fig. 5, except that 0.25- ml reaction mixtures were used. All other conditions were as previously described.

in much lower concentrat,ions than the other methylated com- pounds. This does not agree with the previous analysis (14). It should be emphasized that approximately 50% of the S-RNA we are examining is derived from organisms grown in the absence of methionine and that competition for the last traces of methio- nine may alter the normal distribution of methylated bases. The assumption made in the above comparison of our results and those of Dunn et al. (14) is that S-adenosylmethionine is the only methyl donor for the formation of methylated S-RNA.

The function of methylated bases in S-RNA is presently unknown. Studies from a number of laboratories suggest that the distribution of these bases throughout S-RNA molecules is nonrandom (15-19). The observation that the enzymes which catalyze methylation of S-RNA are species-specific, as well as strain-specific, implies that they impart to the cell a high degree of specificity in the distribution of methylated bases. This suggests that these bases play some role in the function of S-RNA. However, to date, evidence has accumulated which suggests that there is no difference between methylated and methyl-deficient S-RNA in the ability to accept amino acids (20, 21). To test adequately whether the methylated bases are intimately in- volved in the transfer of amino acids by the amino acid activation enzymes, it will be necessary to determine whether, after methyla- tion, highly purified S-RNA specific for a particular amino acid is altered in its acceptor ability.

SUMMARY

1. Six enzymes present in Escherichia coli W have been purified and shown to catalyze the transfer of methyl groups from S- adenosylmethionine to different ribonucleotide residues in soluble ribonucleic acid (S-RNA) by the reaction

S-Adenosylmethionine + S-RNA +

CHa-S-RNA + S-adenosylhomocysteine

2. All of these enzymes are highly specific for S-RNA. A large number of both naturally occurring as well as synthetic RNA preparations do not replace S-RNA as methyl group ac- ceptor. Partial degradation of S-RNA with venom phospho- diesterase has little influence on the ability of S-RNA to act as an acceptor.

3. The extent of methylation with different methylating enzymes differs. It was found that the enzymatic methylation of uracil residues of S-RNA occurs to the greatest degree while the enzymatic methylation of adenine residues occurs to the lowest extent. When the methylation reaction has come to a halt with a particular enzyme, the further addition of more of that enzyme does not restore methylation. In contrast, the further addition of S-RNA leads to a resumption of the methylation reaction.

4. Stepwise degradation of methyl-labeled S-RNA by snake venom phosphodiesterase liberates both radioactivity and ultra- violet-absorbing material at approximately equal rates. These results suggest that there is a random distribution of 14C-methyl groups throughout the chains of the heterogeneous mixture of RNA used.

5. Different combinations of the enzymes, except with two enzymes which both yield the same product, lead to additive incorporation of methyl groups. These results suggest that each enzyme acts independently in catalyzing the methylation of particular sites in S-RNA.

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3482 Enzymatic Methylation of RNA and DNA. IV Vol. 239, No. 10

6. S-Adenosylhomocysteine, one of the presumed products of 11. FLEISSNER, E., AND BOREK, E., Proc. Natl. Acad. Sci. U. S.,

the methyl&ion reaction, is a competitive inhibitor of some of the 48, 1199 (1962).

enzymes. This is particularly true of the enzyme which cata- 12. HURWITZ, J., EVANS, A., BABINET, C., AND SKALKA, A., Cold

lyzes the methylation of uracil residues of S-RNA. The inhibi- Spring Harbor symposia on quantitative biology. Vol. 28, Long Island Biological Association, Cold Spring Harbor,

tion can be reversed by increasing concent,rations of S-adenosyl- New York, 1963, p. 59.

methionine. 13. DOTY, P., BOEDTKER, H., FRESCO, J. R., HASELKORN, R., AND LITT. M.. Proc. Natl. Acad. Sci. U. S.. 45.482 (1959).

1.

2.

3.

4.

5. 6. 7.

8.

9. 10.

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Jerard Hurwitz, Marvin Gold and Monika AndersACID-METHYLATING ENZYMES

IV. THE PROPERTIES OF THE SOLUBLE RIBONUCLEIC The Enzymatic Methylation of Ribonucleic Acid and Deoxyribonucleic Acid:

1964, 239:3474-3482.J. Biol. Chem. 

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