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Chemical modification of viral ribonucleic acid
BIOCHIMICA ET BIOPHYSICA ACTA
217
BBA 8267 C H E M I C A L M O D I F I C A T I O N O F V I R A L R I B O N U C L E I C ACID II. BROMINATION AND IODINATION* K. W. B R A M M E R ' *
Virus Laboratory, University of California, Berkeley, Calif. (U.S.A.) (Received J a n u a r y 29th, 1963)
SUMMARY
The halogenation of tobacco mosaic virus (TMV)-RNA was studied using low levels (molar ratio of reagent:nucleotide was 1:5 and less) of brominating and iodinating agents under conditions in which infectious TMV-RNA itself was stable. The sites of reaction in RNA were the bases, of which only adenine was completely resistant to halogenation. N-Bromosuccinimide reacted with both guanine and cytosine at pH 7 and pH 9 although reaction with guanine was more favored at pH 9. Bromine showed a more specific action, reacting almost exclusively with cytosine at pH 7 and with guanine at pH 9. Although free uridylic acid was readily brominated by both reagents, the uracil residues in RNA were apparently less reactive than either guanine or cytosine. Consequently, very little, if any, bromination of uracil was detected in RNA under the conditions used. The loss of infectivity of TMV-RNA upon reaction with bromine or N-bromosuccinimide indicated that both reagents had a high efficiency of reaction. Using similar mild conditions, iodination of TMV-RNA was not achieved with iodine in potassium iodide solution. N-Iodosuccinimide, although about ten times less efficient than the brominating agents, did inactivate infectious TMV-RNA at pH 7 and o °. Cytosine was the most readily iodinated base under these conditions. Using the reagent labeled with 131I it was found that the binding of 2-3 atoms of iodine per infectious RNA molecule was lethal. The bromination and iodination of guanylic acid and cytidylic acid with these reagents under the conditions used for the halogenation of TMV-RNA provided some evidence regarding the probable nature of the chemical changes which occurred in the macromolecule. Some aspects of these reactions in relation to the mutation of TMV are discussed. INTRODUCTION
Extensive bromination of nucleic acids has been studied by several workers~, 3 and it has been shown that the bases of the nucleic acid represent the most readily " F o r p a p e r i o f t h i s series see ref. i. "* P r e s e n t a d d r e s s : B i o c h e m i c a l R e s e a r c h D e p a r t m e n t , Pfizer, Ltd., Sandwich, K e n t (Great Britain).
Biochim. Biophys. Acta, 72 (1963) 217-229
218
K . W . BRAMMER
brominated sites in the molecule. Of the bases which are normally found in DNA and RNA, only adenine was resistant to bromination. JONES AND WOODHOUSE2 performed base analyses on extensively brominated DNA and found that thymine was considerably less brominated than guanine and cytosine and that adenine had not reacted. KANNGIESSER3 reported the identification of 5-bromouracil, 5-bromocytosine and 8-bromoguanine in acidic hydrolysates of brominated RNA. Iodination of RNA is much more difficult to achieve than bromination and no detailed report of this reaction was found in the literature. It was of particular interest to investigate the reaction with N-bromosuccinimide since its reaction with the infectious TMV-RNA was shown in this laboratory to induce mutations. The level of mutation was considerably lower than that obtained by reaction with nitrous acid, but numerous mutants were isolated and some were shown to have changes in the amino acid composition of the isolated protein as compared with that of normal TMV4,5. The present paper describes experiments designed to study the preferred sites and the mechanisms of halogenation of TMV-RNA with bromine, N-bromosuccinimide and N-iodosuccinimide. The properties desired of the reaction were chemical specificity for reaction with a single base and mutagenic action towards TMV-RNA. A study of the chemistry of the N-bromosuccinimide reaction was, therefore, of particular interest. Since the level of reaction most likely to give rise to a mutation was of the order of I halogenation per RNA chain (6 400 nucleotides) it was desirable to use low levels of reagent. The availability of 1311 made studies of the iodination of TMV-RNA possible at very low levels of reaction.
METHODS
Infectious TMV-RNA was prepared by a modification of the phenol method in the presence of bentonite e. Non-infectious TMV-RNA (obtained as a by-product during the preparation of TMV-protein with 67% acetic acid) was used for the chemical studies on the halogenation reactions. This RNA was freed from protein by the phenol procedure 7. The reactions of TMV-RNA at pH 7 were carried out in lO -3 M EDTA buffer and those at pH 9 in lO -1 or IO-z M carbonate-bicarbonate solution. The halogenated RNA was normally freed from excess reagent by precipitating it twice with 2 vol. of absolute ethanol at o °'. When radioactive reagents were used the RNA was precipitated 4-6 times. Halogenated infectious TMV-RNA preparations were reconstituted with TMV-protein in pyrophosphate solution s before assaying for infectivity. Nucleotide mixtures of the 2'- and 3'-isomers obtained from Pabst Laboratories were used as model substrates for studying the halogenation mechanisms. Both bromination and iodination of the nucleotides was accompanied by a substantial decrease in their ultraviolet absorption and this criterion was used both for detecting reaction and also for comparing the relative rates of reaction of the nucleotides with a particular reagent. Measurements were made on a Cary recording spectrophotometer Model 14 M. Nucleotide analyses were carried out by hydrolysis of the RNA for 18 h at 37 ° " It was Observedthat the brominating action of Brs continued in the 67 ~o alcohol suspension. Biochim. Biophys. Acta, 72 (1963) 217-229
BROMINATION AND IODINATION OF
TMV-RNA
219
with an approx. 3o-fold excess of 0.3 N NaOH. The alkaline hydrolysate was applied directly on to a sheet of Whatman No. 3 MM chromatography paper and chromatographed in the first dimension for 20 h using 70% isopropanol as solvent and having ammonia present in the atmosphere 9. The nucleotides had low RF values in this solvent and only partial separation was achieved, but the main purpose was to disperse the alkali sufficiently to permit development in the second dimension (electrophoresis in 0.05 M ammonium formate (pH 3.5) at 18 V/cm for 3 h) and achieve complete resolution of the nucleotides, without interference. In this way the neutralization and desalting procedures were eliminated. The nucleotide zones were cut out, eluted in o.oi N HC1, and estimated spectrophotometrically. Aqueous solutions of bromine, N-bromosuccinimide and N-iodosuccinimide (approx. I mg/ml) were prepared at o ° and the latter two were used immediately after preparation. The method of preparation of N-iodosuccinimide labeled with 131I was by the addition of freshly prepared, dried silver salt of succinimide (excess) to a solution of iodine (40 rag) containing 131I (0.5 mC) in dry acetone or ether. On stirring, the reaction proceeded by the precipitation of half the iodine as silver iodide while the remaining iodine reacted to form the acetone-soluble N-iodosuccinimide. When the yellow iodine color had disappeared, the reaction was complete and excess silver succinimide and the silver iodide was removed by centrifugation. The supernatant was removed and allowed to evaporate to dryness in a current of air, leaving a pale yellow residue of N-iodosuccinimide. The activity of the iodine present was 8.5" IOs counts/min/mg of iodine. This meant that 25 counts/min were associated with each atom of iodine bound per 6 400 nucleotides in I ml of a solution of 0.05 mg hypochromed RNA (absorbancy i). Due to the 8-day half-life of 131I such a preparation of N-iodosuccinimide was effectively useful for the iodination of TMV-RNA during a period of about 4 weeks after preparation. Prepared in this way, the reagent was not pure and approximately twice the levels were required to produce the same inactivation of TMV-RNA as described for the commercial (non-radioactive) product (see Fig. 5). RESULTS
Bromination with N-bromosuccinimide
The reaction of the separate nucleotides with an equimolar amount of N-bromosuccinimide at pH 7 and o ° was followed b y plotting the decrease in maximum absorbancy of ultraviolet light as a function of time. The results, shown in Fig. I, indicated that there was no reaction with adenylic acid, a fairly slow reaction with uridylic acid and a very rapid reaction with both guanylic and cytidylic acids. A similar reaction pattern was obtained at pH 9 except that the reaction of uridylic acid with N-bromosuccinimide was greatly accelerated (Fig. 2). The level of bromination in these experiments was not sufficient for the complete disappearance of the typical ultraviolet absorption maxima of the original nucleotides. Complete bromination of uridylic and guanylic acids was accompanied by complete disappearance of the typical ultraviolet absorption peaks, but there was a considerable end absorption in the case of guanylic acid. In the case of cytidylic acid, a new peak was produced at 237 m/, simultaneously with the destruction of the normal peak at 270 m#. TMV-RNA was mixed with N-bromosuccinimide at p H 7 (molar ratio of nuBiochim. Biophys. Acta, 72 (1963) 2x7-229
220
K . W . BRAMMER
D
D
o
7o .=:-
.a
6o
I 40
so
eb Time(rnin)
Fig. i. Reaction of nucleotides with N-bromosuccinimide at pH 7. Nucleotides were mixed with reagent in the molar ratio i : i and aliquots of solution withdrawn after fixed times for ultraviolet reading. The actual solutions prepared contained --~IO/~moles/ml of each component. 5° X nucleotide solution Was mixed with 5° ;1, of N-bromosuccinimide solution and io ~ of lO-2 M EDTA (pH 7.o). AMP ([~--N), GMP (O---O), CMP (LX--A) and UMP ( H ) -
N u c l e o t i d e a n a l y s e s w e r e c a r r i e d o u t b y h y d r o l y s i s of t h e R N A for 18 h a t 37 ° c l e o t i d e : N - b r o m o s u c c i n i m i d e , 5:1) a n d m a i n t a i n e d at o ° for I h. A f t e r t w o prec i p i t a t i o n s w i t h e t h a n o l t h e b r o m i n a t e d R N A was a n a l y z e d for n u c l e o t i d e s (see T a b l e I). Two comparable experiments were carried out at pH 9 using nucleotide: Nb r o m o s u c c i n i m i d e m o l a r r a t i o s of I O : I a n d 5 :I, r e s p e c t i v e l y , a n d t h e n u c l e o t i d e r a t i o s o f b o t h t h e s e s a m p l e s are also s h o w n in T a b l e I. T h e a m o u n t s o f g u a n y l i c , u r i d y l i c a n d c y t i d y l i c acids are r e l a t e d to a d e n y l i c acid, w h i c h r e m a i n s c o n s t a n t d u r i n g t h e
I00~
80
6o 4ok
"6
2o 0
I
0
I
I
10 20 Time (min)
30
40
Fig. 2. Reaction of uridylic acid with N-bromosuccinimide at pH 7 ( 0 - - 0 ) and 9 (O---O). (Conditions as in Fig. i ; pH 9 buffer was carbonate-bicarbonate, io -j M). Biochim. Biophys. Acta, 72 (1963) 217-229
221
BROMINATION AND IODINATION O F T M V - R N A TABLE I REACTION OF T M V - R N A
WITH N-BROMOSUCCINIMIDE
(NBSI) AT
P H 7 AND 9
Nucleotid* ratios Adenylic acid
TMV-RNA After b r o m i n a t i o n at p H 7, nucleotide: N B S I 5 : I After b r o m i n a t i o n at p H 9, nucleotide: N B S I io: i nucleotide: N B S I 5 : i
Guanylic a c i d
Uridylic acid
Cytidylic acid
I
0.87
o.91
0.66
i
0.76
0.88
0.56
i i
0.79 0.65
0.90 o.87
0.62 0.56
reaction. Each set of figures represents the average of three separate determinations and the maximum error associated with each figure is 2 %. In the reaction with N-bromosuccinimide at both pH 7 and 9, guanine and cytosine represent the most reactive sites. At pH 7 the proportion of each decreases by about 15%, whereas, at a similar level of N-bromosuccinimide at pH 9, the guanine content decreases 25 % and the cytosine about 15 %. The change in uridylic acid content is significant, but only just detectable by this method. The brominated infectious TMV-RNA from which mutants were isolated was obtained by reaction with N-bromosuccinimide at pH 7, so it appears that the mutagenic event might have been either due to a change in cytosine or guanine. Reaction with bromine
Using the same conditions as for the reaction with N-bromosuccinimide the rate of reaction of the separate nucleotides with bromine was similarly studied. The 100
gO
.= o
,....(
8(3
~°o
2'0
4'0
Time (rnin)
do
do
Fig. 3. Reaction of guanylic acid w i t h b r o m i n e at p H 7 ( O - - O ) and 9 ( 0 - - - 0 ) . Nucleotide concentration, , ~ i o /*moles/ml. B r o m i n e concentration, ~ 5 /,moles/ml. 5 ° ;t of each mixed in presence of :o 2 of buffer.
reaction with the pyrimidine nucleotides was almost instantaneous at both pH 7 and 9. Bromination of cytidylic acid resulted in the formation of the new ultraviolet absorption peak at 237 m/z in each case. The reaction of guanylic acid with bromine was considerably faster at pH 9 than at pH 7 (Fig. 3). Once again adenylic acid was completely stable to bromination. Biochim. Biophys. Acta, 72 (1963) 217-229
222
K.W.
BRAMMER
The change in the nucleotide composition of RNA after treatment with bromine (I mole Brz:io moles nucleotide) was studied at pH 7 and 9, aliquots of the brominated RNA being taken after 15 min and 2 h. The results obtained are shown in Table II. These figures indicate that at pH 7 and using a low level of bromine, only cytosine in the RNA is attacked. At pH 9 the specificity is quite different and guanine
TABLE REACTION OF T M V - R N A
II
WITH BROMINE AT P H 7 AND 9
Nuvleotide ratios
TMV-RNA B r o m i n a t i o n a t p H 7, nucleotide-Br v io:i 15 r a i n 2 h B r o m i n a t i o n a t p H 9, nucleotide--Br v io:i 15 m i n 2 h
Adenylic acid
Guanylic acid
Uridylic acid
Cytidylic acid
i
0.88
0.92
o.66
i I
0.88 0.87
0.92 o.91
0.56 0.53
i i
o.86 o.81
o.89 0.92
0.66 0.66
seems the only base brominated. This change in specificity is much more dramatic than was obtained using N-bromosuccinimide, but with that reagent too, the bromination of guanine appeared to be favored at pH 9.
Bromination products of cytidylic and guanylic acids The foregoing experiments indicated that the guanine and cytosine residues in RNA were the favored sites of bromination using fairly low levels of brominating agent at either pH 7 or 9. An attempt was made to identify the products of bromination of cytidylic and guanylic acids under these conditions. Cytidylic acid: The bromination of uridylic acid, uridine, and other uracil derivatives has been studied in detail by several workers 1°-1~, and it was established that at pH 7 one mole equivalent of bromine was absorbed and caused complete loss of the characteristic uracil ultraviolet absorption peak. This was due to saturation of the 5,6 ethylenic linkage in uracil by the formation of a 5-bromo-6-hydroxyhydrouracil d'erivative 11,12. The latter compound was unstable and either heating or exposure to acidic pH resulted in the reappearance of an ultraviolet absorption spectrum due to elimination of water from the 5,6 position forming a 5-bromouracil derivative. This compound could then react with another mole equivalent of bromine to form a 5,5dibromo-6-hydroxyhydrouracil derivative. The present work has shown that the bromination of cytidylic acid proceeds in a similar manner. The first bromination product of cytidylic acid using bromine or N-bromosuccinimide at either pH 7 or 9 had an absorption maximum at 237 m#. This was the s a m e ~.max as found for 5,6-dihydrocytosine derivatives is and appeared to be B i o c h i m . B i o p h y s . A c t a , 72 (1963) 2 1 7 - 2 2 9
BROMINATION AND IODINATION OF TMV-RNA
22 3
characteristic for derivatives of cytosine in which the 5,6 ethylenic linkage was saturated. Provided that excess brominating agent was absent, the action of heat or acid on this brominated derivative resulted in the loss of the ultraviolet absorption peak at 2,37 m/z and the formation of a new peak at 287 m/~. Further bromination resulted irr the disappearance of the latter peak and a compound having an absorption peak at 237 m# was again produced. The same sequence of reactions as proposed for uracil derivatives by WANGTM and MOORE AND ANDERSONn apparently took place with cytidylic acid. That is, NHz
NH,
I
I
/tiC\ N C--H
I
I
C--H
I
R
>
•
NH,
I
, / C \ --H N C~,B r
Br, H,O
NH,
Heat
|
/C~IH
°rH+
I
÷
I
I
.C
C--H
I
R
I
/tiC\ N C--Br
R
//C\ Br N C~B r
Br, H20
>
I
I
H
]
R
Cytidylic acid, 5-Bromo-6-hydroxy- 5-Bromocytfdylic acid, 5,5-Dibromo-6-hydroxy~.max (H,O) ~= 287 m/~. hydrocytidylic acid, ~max (HzO) = 270 m#. hydrocytidylic acid, ~max (H,O) 237 rn/~. )lmax (H20) = 237 m/~.
Saturation of the 5,6 ethylenic linkage in cytosine derivatives still leaves the
J
f
conjugated chromophore --C ---- N -- C = O to which the absorption peak at 237 m/z is attributed. The structure of the 5,6-saturated uracil derivatives in the favored keto form does not have this chromophore, nor do they have an absorption peak above 21o m/z. 5-Bromocytidylic acid was readily separable from cytidylic acid on electrophoresis at pH 3.5 under the standard conditions for nucleotide separation. The mobility of 5-bromocytidylic acid was o.81 relative to that of uridylic acid (very similar to guanylic acid), whereas cytidylic acid had a mobility of 0.20 relative to uridylic acid. When bromination mixtures of cytidylic acid with either bromine or N-bromosuccinimide were separated directly by electrophoresis at pH 3.5, cytidylic acid and 5-bromocytidylic acid were the only compounds detectable by absorption of ultraviolet light. This indicated that 5-bromo-6-hydroxyhydrocytidylic acid was rapidly converted to 5-bromocytidylic acid at pH 3.5. It was shown that this conversion took place within 5 h at room temperature at any pH less than 6. The following were the ultraviolet absorption characteristics for 5-bromocytidylic acid, the emax being the absorbancy per mole of phosphorus: in water, emax 6800; ~max 287 m/~; in o.oi N HC1, emax 9000; ~.max 298 m/~. When 5-bromo-6-hydroxyhydrocytidylic acid was set aside at room temperature and alkaline p H (pH 8-I3), the absorption peak at 237 m/z disappeared with the formation of a new peak at about 29o mp in water. The reaction was very slow at pH 8 but was complete within 2 h at pH 13. The following properties established that it was not 5-bromocytidylic acid: in water, emax 7500; ~max 287 m/z; in o.oi N HC1, emax 9300; Amax 3Ol mp; in o.oi N NaOH, emax 7oo0; Amax 316 rap. The mobility of the compound on electrophoresis at pH 3.5 was 0.44 compared Biochim. Biophys. Acta, 72 (1963) 217-229
224
K. W. BRAMMER
with uridylic acid. This was similar to the mobility of adenylic acid and considerably less than that of 5-bromocytidylic acid (o.81). Both cytidylic and 5-bromocytidylic acids have a similar ~max in water and in alkali, whereas there was a difference of about 30 m # in this case. FUKUHARA AND VISSER14 probably obtained the nucleoside derivative of this same compound by passing a solution of 5-bromo-6-hydroxyhydrocytidine through a column of a basic resin in the O H - form. They recorded emax 7837, ~tmax 292 m/~, for the compound at p H 7 and presented evidence suggesting that it was 5-hydroxycytidine. These experiments concerning the stability of the first bromination product of cytidylic acid indicated that it was necessary to incubate RNA after bromination at a mildly acidic p H to ensure that bromination of cytosine resulted in formation of the relatively stable 5-bromocytosine. Guanylic acid: The bromination of guanylic acid was not studied in such detail, but a derivative was obtained which absorbed ultraviolet light on treatment of guanylic acid with bromine at p H 7. The spectrum in acid had a slight hump at about 280 in/z, but not as pronounced as that of guanylic acid itself. In alkali the spectra of both guanylic acid and the brominated derivative had a single peak, but at a different wavelength. A comparison of the ultraviolet absorption characteristics of these compounds is shown in Table I I I . TABLE
III
C O M P A R I S O N OF U L T R A V I O L E T A B S O R P T I O N P R O P E R T I E S OF G U A N Y L I C ACID A N D B R O M I N A T E D
Guanyl i c acid Medium
o.oI N HCI o.oi N NaOH
GUANYLIC ACID
B r o m i n a t e d guanylic acid . . . . . . . . . . .
*ma*
~max (ma)
e,n~,
~. . . . {into
12 2oo I I IOO
257 258-263
9ooo 74oo
26o 27o
The distinct similarities in ultraviolet absorption spectra of the brominated derivative and parent compound suggested that they were still closely related structurally. I t was more likely that the bromination was a substitution reaction rather than an addition. It appeared probable that this was the 8-bromoguanine derivative as identified in acid hydrolysates of brominated RNA by KANNGIESSER3. The electrophoretic mobility of the brominated compound at p H 3.5 was slightly less (0.76) than that of guanylic acid (o.81). The same compound was apparently produced when guanylic acid was treated with less than I mole equivalent of N-bromosuccinimide at p H 7, but further bromination to non-ultraviolet-absorbing products occurred when excess of the reagent was present. The bromination of guanylic acid at p H 9 using either bromine or Nbromosuccinimide appeared to follow a different course since in neither case was a compound formed which absorbed ultraviolet light.
Iodination with N-iodosuccinimide The relative rates of iodination of the separate nucleotides at p H 7 and o ° were determined b y comparing the rates of decrease in ultraviolet absorption as previously Biochira. B i o p h y s . A c t a , 72 (1963) 2 1 7 - 2 2 9
BROMINATION AND IODINATION OF T M V - R N A
225
described for the bromination experiments. Reaction was detected only with cytidylic and uridylic acids (Fig. 4). The iodination of cytidylic acid was a much faster reaction than the iodination of uridylic acid under the same conditions. The extent of reaction was limited b y the decomposition of N-iodosuccinimide in aqueous solution which liberated free iodine.
100,
90
@
8o
o x•
70
E •~
60
"6
5%
io
go
15o
1~o
Time (min)
Fig. ¢. Reaction of nucleotides with N-iodosuccinimide at pH 7.0. (Conditions as in Fig. i). AMP and GMP ( A - - A ) , CMP (0---0) and UMP ( O - - O ) .
When a similar series of reactions was conducted at p H 9, uridylic acid was the only nucleotide in which a decrease in ultraviolet absorption was detected and in this case the decrease was only slight (about 5 % in I h). One of the factors influencing this diminished reactivity of N-iodosuccinimide was probably the faster decomposition of the reagent in aqueous solution at p H 9 compared with p H 7. Since cytidylic acid was clearly the most rapidly iodinated nucleotide at p H 7, a more detailed study of this reaction was made. Cytidylic acid was treated with 2 mole equivalents of N-iodosuccinimide in lO -3 M E D T A (pH 7) at o ° for I h. Iodine and other ether-soluble products were removed b y four ether extractions and the aqueous phase was concentrated and investigated by paper electrophoresis at p H 3.5 for 3 h. Markers of the four nucleotides were also applied and separated. The iodination mixture contained two components which absorbed ultraviolet light, one having a low mobility which was shown to be unreacted cytidylic acid, while the other had a mobility of o.81 compared to uridylic acid. This was the same mobility as guanylic acid and also the same as that found previously for 5-bromocytidylic acid. This zone was eluted in water and was shown to have the following ultraviolet absorption characteristics, based on phosphorus analysis: in water, emax 5800; ~max 292 m/~; in o.oi N HC1, emax 7700; ;tmax 307 In#. A comparison of these figures with the corresponding values determined for 5bromocytidylic acid shows that the emax values of the iodinated derivative are slightly lower and that the absorption maxima occur at slightly higher wavelengths than those of the brominated derivative. The behavior of the ultraviolet absorption spectra Biochim. Biophys. Aaa, 72 (1963) 217-229
226
K. W. BRAMMER
on acidification is similar with both compounds. These similarities of spectra, together with the fact that both compounds exhibit the same mobility on electrophoresis at p H 3.5 (indicative of the same p K value) led to the conclusion that the iodinated derivative was 5-iodocytidylic acid. Infectious TMV-RNA was treated with inactivating levels of N-E131I]iodosuccinimide at p H 7 for I h and after 6 precipitations of the iodinated RNA with ethanol the number of atoms of iodine bound per 6400 nucleotides was calculated for the various samples. Aliquots of these same samples were reconstituted with TMVprotein and assayed for infectivity. The results of several such experiments (Table IV) indicated that the binding of about 4 atoms of iodine per 6400 nucleotides in the total RNA population was necessary to cause the infectivity of the RNA to be reduced to 37% of the control RNA (i.e., the inactivation produced by one lethal hit). TABLE I N A C T I V A T I O N OF I N F E C T I O U S T M V - R N A
Atoms of I #er 6400 nucleolide8 o 0.5 1.2 2.3 2.5 3.5 4.3 5.5 12. 7
IV CAUSED BY B I N D I N G OF I O D I N E
Infeaivity (% of control) IOO 9o ~IOO 90 65 5° 4° 4 0. 5
For investigation of the actual iodination site in the RNA a sample of noninfectious RNA was iodinated with a higher level (approx. I mole reagent: 2 moles nucleotide) of the N-E131I~iodosuccinimide and then precipitated with ethanol until the ratio of counts/min to absorbancy was constant. In this preparation about 84 atoms of iodine were bound per 6400 nucleotides. The iodinated RNA was degraded and the products separated on paper in two ways. (a) By alkaline hydrolysis to the nucleotides and separation of the nucleotide mixture b y the method combining paper chromatography and p H 3.5 electrophoresis as described previously. (b) By total digestion to nucleosides using the mixture of phosphodiesterases and phosphomonoesterases present in crude snake venom. The nucleosides were then separated on paper using the 2-dimensional system of FELIX et al. ~. Both papers were radioautographed and in each case most of the radioactivity (but no ultraviolet-absorbing material) remained at the origin indicating that considerable decomposition of the iodinated derivative had occurred during the digestion procedure. The only other radioactive area present on the nucleotide paper was associated with the guanylic acid zone. This was the expected position for 5-iodocytidylic acid as determined previously. The nucleoside paper also had only one radioactive area besides the origin and Biochim. Biophys. Acta, 72 ( 1 9 6 3 ) 2 1 7 - 2 2 9
BROMINATION AND IODINATION OF TMV-RNA
227
this was located in a position between the purine and pyrimidine nucleosides. A marker of 5-iodocytidine chromatographed in the same 2-dimensional system had a similar position relative to the normal nucleosides. Difficulty was experienced in identifying compounds by their RF values in this system since mobilities in both solvents were sensitive to temperature change and the degree of saturation of the atmosphere in the chromatography cabinet. The evidence of this experiment, together with that from the reaction rate studies on the separate nucleotides, suggested strongly that cytosine was the RNA component most readily iodinated by N-iodosuccinimide at pH 7. Insufficient counts remained on the chromatograms to permit further establishment of the identity of the iodinated compound.
Iodination using iodine in K I solution No change in the ultraviolet absorption spectra of the nucleotides was detected on treatment with iodine in potassium iodide solution at o ° at either pH 7 or 9. A slow decrease in the ultraviolet absorption maximum was detected, however, upon iodination of uridylic acid at 37 ° in carbonate-bicarbonate buffer (lO -2 M) at pH 8-1o. This decrease was more rapid at pH IO than 8 but in no case was the final absorption less than 80% of the original, although free iodine was still present. Iodination of infectious TMV-RNA was attempted in o.oi-o.I M carbonatebicarbonate buffers varying in pH from 8 to 9.5 at 37 °, but in no case was there any inactivation beyond that of the control RNA. When experiments were carried out under the same conditions with 181Ino evidence of the binding of iodine was obtained. In many of these experiments considerable inactivation of infectious TMV-RNA was brought about by the conditions of reaction so it was concluded that iodine itself was not a sufficiently reactive iodinating agent to be of any value in these studies.
The relative efficiency of the halogenating reagents for reaction with TMV-RNA The inactivation of infectious TMV-RNA using different levels of halogenation reagent was studied. All experiments were performed at o ° for I h after which the modified RNA was twice precipitated with ethanol, then reconstituted with TMVprotein and assayed for infectivity. The' reaction with N-bromosuccinimide and N2
"E 100
8 8O
:>_- 60 .~ 40 ._=o, .£_ 20 E ,,. o
100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 700 8 0 0 9 0 0 Moles r e a g e n t added per mole RNA
1000
Fig. 5- Inactivation of TMV-RNA by halogenation reagents. Br s at pH 7 ( / k - - A ) , Bra at p H 9 ( A - - A ) , N-bromosuccinimide a t pH 7 (O--O) and N-iodosuccinimide at p H 7 ( G - - O ) . Biochim. Biophys. Acta, 72 (1963) 2x7-220
228
K. W. BRAMMER
iodosuccinimide were studied at pH 7 and the reaction with bromine at pH 7 and 9. The relative efficiency of bromine and N-bromosuccinimide for the inactivation of TMV-RNA at pH 7 are quite similar. Bromine at pH 9 is significantly less efficient. The iodination using N-iodosuccinimide at pH 7 is a much less efficient inactivating reaction requiring approximately a Io-fold excess of N-iodosuccinimide compared with the levels of brominating agent needed to produce similar inactivation under the same conditions (Fig. 5). DISCUSSION
From the point of view of finding reactions which chemically modify TMV-RNA, preferably by reacting specifically with only one of the bases, both bromination and iodination under the correct conditions are of interest. The only halogenation reagent wich has been established as definitely mutagenic towards TMV is N-bromosuccinimide acting at pH 7. The present work shows that this leads most probably to bromination of guanine or cytosine (or both). Since it is not known whether only one, both or neither of these events causes the mutations, correlation of the reaction with the RNA to the change induced in the mutant protein is made difficult. It is of interest to note, however, that the changes in amino acid composition of mutant proteins induced by this reaction show marked similarities to those in some mutants obtained by the action of nitrous acid on infectious TMVRNA 4. Bromine itself is shown to produce more selective bromination than N-bromosuccinimide. Low levels of reaction with RNA at pH 7 cause almost exclusive bromination of cytosine, whereas, at pH 9 tt:e reaction is similarly specific for the bromination of guanine. After bromination at pH 7 it is desirable to expose the modified RNA to mildly acidic conditions before reconstitution to favor the formation of the relatively stable 5-bromocytosine rather than other products. No informatizn per~aining to the nature of the products obtained on reaction of guanine derivative; with bromine at pH 9 is available at present. Bromination of guanine compounds at pH 7 probably results in formation of the 8-bromoguanine derivative. It has not yet been established whether the reactions of bromine with TMV-RN~', at pH 7 and/or 9 lead to mutation of the virus*. If only one of these reactions leads to mutation, it might be possible to determine the nature of the mutagenic change which occurs on reaction with N-bromosuccinimide. In any case it would be of interest to make a comparison of the amino acid composition of mutant proteins produced by different bromination reactions. The reaction of TMV-RNA with N-iodosuccinimide at pH 7 favors the iodination of cytosine. This reaction does not appear to be mutagenic. It is possible that the iodine is too large an atom to cause a mutagenic change and is only inactivating. The data in Table IV indicates that the binding of about 4 atoms of iodine per 64oo nucleotides is necessary to produce the inactivation of one lethal hit. Since the RNA preparations used consist of only 50-80% of full-length, infectious molecules, the
* A conclusive s t u d y of the action on TMV of m u t a g e n s of low efficiency is at p r e s e n t n o t possible in our laboratory. This is due to the u n u s u a l l y high frequency of w h a t a p p e a r to be s p o n t a n e o u s m u t a t i o n s observed during the p a s t year in our greenhouse.
Biochim. Biophys. Acta, 72 (1963) 217-220
BROMINATION AND IODINATION OF
TMV-KNA
229
number of iodinations which cause loss of infectivity is 2-3 per infective RNA molecule. This is similar to the number of alkylations necessary to produce the same inactivation of TMV-RNA 1. ACKNOWLEDGEMENTS
The author expresses his thanks to Dr. H. FRAENKEL-CONRATfor his advice and helpful discussion during the course of this work and for making the laboratory facilities available, and to Mrs. B. SINGER for discussion and advice on practical techniques. This investigation was supported by a grant from the National Science Foundation. REFERENCES I H. FRAENKEL-CONRAT,Biochim. Biophys. Acta, 49 (1961) 169. i A. S. JONES AND D. L. WOODHOUSE,Nature, 183 (1959) 16o 3. 8 W. KANNGIESSER, Z. Physiol. Chem., 316 (1959) 146. 4 A . TSUGITA AND H. FRAENKEL-CONRAT, J. Mol. Biol., 4 (1962) 736 A. TSUGITA, J. Mol. Biol., 5 (1962) 284. t H. FRAENKEL-CONRAT,B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54" 7 A. GIERER AND G. SCHRAMM, Z. Naturforsch., I I b (1956) 138. s H. FRAENKEL~CONRAT AND B. SINGER, Biochim. Biophys. Aaa, 33 (1959) 359. o R. MAEKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 552. to W . E. COHN, Biochem. J., 64 (1956) 28. 11 A. M. MOORE AND S. M. ANDERSON, Can. J. Chem., 37 (1959) 590. ts S. Y. WANG, Nature, 18o (1957) 91. is D. SHUGAR, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol., 3 196o, p. 53. 14 T. K. FUKUHARA AND D. W. VISSER, Biochemistry, I (1962) 563 . 15 F. FELIX, J. L. POTTER AND M. LASKOWSKI, f . Biol. Chem., 235 (196o) II5O.
Biochim. Biophys. Acta, 72 (1963) 2 1 7 - 2 2 9
217
BBA 8267 C H E M I C A L M O D I F I C A T I O N O F V I R A L R I B O N U C L E I C ACID II. BROMINATION AND IODINATION* K. W. B R A M M E R ' *
Virus Laboratory, University of California, Berkeley, Calif. (U.S.A.) (Received J a n u a r y 29th, 1963)
SUMMARY
The halogenation of tobacco mosaic virus (TMV)-RNA was studied using low levels (molar ratio of reagent:nucleotide was 1:5 and less) of brominating and iodinating agents under conditions in which infectious TMV-RNA itself was stable. The sites of reaction in RNA were the bases, of which only adenine was completely resistant to halogenation. N-Bromosuccinimide reacted with both guanine and cytosine at pH 7 and pH 9 although reaction with guanine was more favored at pH 9. Bromine showed a more specific action, reacting almost exclusively with cytosine at pH 7 and with guanine at pH 9. Although free uridylic acid was readily brominated by both reagents, the uracil residues in RNA were apparently less reactive than either guanine or cytosine. Consequently, very little, if any, bromination of uracil was detected in RNA under the conditions used. The loss of infectivity of TMV-RNA upon reaction with bromine or N-bromosuccinimide indicated that both reagents had a high efficiency of reaction. Using similar mild conditions, iodination of TMV-RNA was not achieved with iodine in potassium iodide solution. N-Iodosuccinimide, although about ten times less efficient than the brominating agents, did inactivate infectious TMV-RNA at pH 7 and o °. Cytosine was the most readily iodinated base under these conditions. Using the reagent labeled with 131I it was found that the binding of 2-3 atoms of iodine per infectious RNA molecule was lethal. The bromination and iodination of guanylic acid and cytidylic acid with these reagents under the conditions used for the halogenation of TMV-RNA provided some evidence regarding the probable nature of the chemical changes which occurred in the macromolecule. Some aspects of these reactions in relation to the mutation of TMV are discussed. INTRODUCTION
Extensive bromination of nucleic acids has been studied by several workers~, 3 and it has been shown that the bases of the nucleic acid represent the most readily " F o r p a p e r i o f t h i s series see ref. i. "* P r e s e n t a d d r e s s : B i o c h e m i c a l R e s e a r c h D e p a r t m e n t , Pfizer, Ltd., Sandwich, K e n t (Great Britain).
Biochim. Biophys. Acta, 72 (1963) 217-229
218
K . W . BRAMMER
brominated sites in the molecule. Of the bases which are normally found in DNA and RNA, only adenine was resistant to bromination. JONES AND WOODHOUSE2 performed base analyses on extensively brominated DNA and found that thymine was considerably less brominated than guanine and cytosine and that adenine had not reacted. KANNGIESSER3 reported the identification of 5-bromouracil, 5-bromocytosine and 8-bromoguanine in acidic hydrolysates of brominated RNA. Iodination of RNA is much more difficult to achieve than bromination and no detailed report of this reaction was found in the literature. It was of particular interest to investigate the reaction with N-bromosuccinimide since its reaction with the infectious TMV-RNA was shown in this laboratory to induce mutations. The level of mutation was considerably lower than that obtained by reaction with nitrous acid, but numerous mutants were isolated and some were shown to have changes in the amino acid composition of the isolated protein as compared with that of normal TMV4,5. The present paper describes experiments designed to study the preferred sites and the mechanisms of halogenation of TMV-RNA with bromine, N-bromosuccinimide and N-iodosuccinimide. The properties desired of the reaction were chemical specificity for reaction with a single base and mutagenic action towards TMV-RNA. A study of the chemistry of the N-bromosuccinimide reaction was, therefore, of particular interest. Since the level of reaction most likely to give rise to a mutation was of the order of I halogenation per RNA chain (6 400 nucleotides) it was desirable to use low levels of reagent. The availability of 1311 made studies of the iodination of TMV-RNA possible at very low levels of reaction.
METHODS
Infectious TMV-RNA was prepared by a modification of the phenol method in the presence of bentonite e. Non-infectious TMV-RNA (obtained as a by-product during the preparation of TMV-protein with 67% acetic acid) was used for the chemical studies on the halogenation reactions. This RNA was freed from protein by the phenol procedure 7. The reactions of TMV-RNA at pH 7 were carried out in lO -3 M EDTA buffer and those at pH 9 in lO -1 or IO-z M carbonate-bicarbonate solution. The halogenated RNA was normally freed from excess reagent by precipitating it twice with 2 vol. of absolute ethanol at o °'. When radioactive reagents were used the RNA was precipitated 4-6 times. Halogenated infectious TMV-RNA preparations were reconstituted with TMV-protein in pyrophosphate solution s before assaying for infectivity. Nucleotide mixtures of the 2'- and 3'-isomers obtained from Pabst Laboratories were used as model substrates for studying the halogenation mechanisms. Both bromination and iodination of the nucleotides was accompanied by a substantial decrease in their ultraviolet absorption and this criterion was used both for detecting reaction and also for comparing the relative rates of reaction of the nucleotides with a particular reagent. Measurements were made on a Cary recording spectrophotometer Model 14 M. Nucleotide analyses were carried out by hydrolysis of the RNA for 18 h at 37 ° " It was Observedthat the brominating action of Brs continued in the 67 ~o alcohol suspension. Biochim. Biophys. Acta, 72 (1963) 217-229
BROMINATION AND IODINATION OF
TMV-RNA
219
with an approx. 3o-fold excess of 0.3 N NaOH. The alkaline hydrolysate was applied directly on to a sheet of Whatman No. 3 MM chromatography paper and chromatographed in the first dimension for 20 h using 70% isopropanol as solvent and having ammonia present in the atmosphere 9. The nucleotides had low RF values in this solvent and only partial separation was achieved, but the main purpose was to disperse the alkali sufficiently to permit development in the second dimension (electrophoresis in 0.05 M ammonium formate (pH 3.5) at 18 V/cm for 3 h) and achieve complete resolution of the nucleotides, without interference. In this way the neutralization and desalting procedures were eliminated. The nucleotide zones were cut out, eluted in o.oi N HC1, and estimated spectrophotometrically. Aqueous solutions of bromine, N-bromosuccinimide and N-iodosuccinimide (approx. I mg/ml) were prepared at o ° and the latter two were used immediately after preparation. The method of preparation of N-iodosuccinimide labeled with 131I was by the addition of freshly prepared, dried silver salt of succinimide (excess) to a solution of iodine (40 rag) containing 131I (0.5 mC) in dry acetone or ether. On stirring, the reaction proceeded by the precipitation of half the iodine as silver iodide while the remaining iodine reacted to form the acetone-soluble N-iodosuccinimide. When the yellow iodine color had disappeared, the reaction was complete and excess silver succinimide and the silver iodide was removed by centrifugation. The supernatant was removed and allowed to evaporate to dryness in a current of air, leaving a pale yellow residue of N-iodosuccinimide. The activity of the iodine present was 8.5" IOs counts/min/mg of iodine. This meant that 25 counts/min were associated with each atom of iodine bound per 6 400 nucleotides in I ml of a solution of 0.05 mg hypochromed RNA (absorbancy i). Due to the 8-day half-life of 131I such a preparation of N-iodosuccinimide was effectively useful for the iodination of TMV-RNA during a period of about 4 weeks after preparation. Prepared in this way, the reagent was not pure and approximately twice the levels were required to produce the same inactivation of TMV-RNA as described for the commercial (non-radioactive) product (see Fig. 5). RESULTS
Bromination with N-bromosuccinimide
The reaction of the separate nucleotides with an equimolar amount of N-bromosuccinimide at pH 7 and o ° was followed b y plotting the decrease in maximum absorbancy of ultraviolet light as a function of time. The results, shown in Fig. I, indicated that there was no reaction with adenylic acid, a fairly slow reaction with uridylic acid and a very rapid reaction with both guanylic and cytidylic acids. A similar reaction pattern was obtained at pH 9 except that the reaction of uridylic acid with N-bromosuccinimide was greatly accelerated (Fig. 2). The level of bromination in these experiments was not sufficient for the complete disappearance of the typical ultraviolet absorption maxima of the original nucleotides. Complete bromination of uridylic and guanylic acids was accompanied by complete disappearance of the typical ultraviolet absorption peaks, but there was a considerable end absorption in the case of guanylic acid. In the case of cytidylic acid, a new peak was produced at 237 m/, simultaneously with the destruction of the normal peak at 270 m#. TMV-RNA was mixed with N-bromosuccinimide at p H 7 (molar ratio of nuBiochim. Biophys. Acta, 72 (1963) 2x7-229
220
K . W . BRAMMER
D
D
o
7o .=:-
.a
6o
I 40
so
eb Time(rnin)
Fig. i. Reaction of nucleotides with N-bromosuccinimide at pH 7. Nucleotides were mixed with reagent in the molar ratio i : i and aliquots of solution withdrawn after fixed times for ultraviolet reading. The actual solutions prepared contained --~IO/~moles/ml of each component. 5° X nucleotide solution Was mixed with 5° ;1, of N-bromosuccinimide solution and io ~ of lO-2 M EDTA (pH 7.o). AMP ([~--N), GMP (O---O), CMP (LX--A) and UMP ( H ) -
N u c l e o t i d e a n a l y s e s w e r e c a r r i e d o u t b y h y d r o l y s i s of t h e R N A for 18 h a t 37 ° c l e o t i d e : N - b r o m o s u c c i n i m i d e , 5:1) a n d m a i n t a i n e d at o ° for I h. A f t e r t w o prec i p i t a t i o n s w i t h e t h a n o l t h e b r o m i n a t e d R N A was a n a l y z e d for n u c l e o t i d e s (see T a b l e I). Two comparable experiments were carried out at pH 9 using nucleotide: Nb r o m o s u c c i n i m i d e m o l a r r a t i o s of I O : I a n d 5 :I, r e s p e c t i v e l y , a n d t h e n u c l e o t i d e r a t i o s o f b o t h t h e s e s a m p l e s are also s h o w n in T a b l e I. T h e a m o u n t s o f g u a n y l i c , u r i d y l i c a n d c y t i d y l i c acids are r e l a t e d to a d e n y l i c acid, w h i c h r e m a i n s c o n s t a n t d u r i n g t h e
I00~
80
6o 4ok
"6
2o 0
I
0
I
I
10 20 Time (min)
30
40
Fig. 2. Reaction of uridylic acid with N-bromosuccinimide at pH 7 ( 0 - - 0 ) and 9 (O---O). (Conditions as in Fig. i ; pH 9 buffer was carbonate-bicarbonate, io -j M). Biochim. Biophys. Acta, 72 (1963) 217-229
221
BROMINATION AND IODINATION O F T M V - R N A TABLE I REACTION OF T M V - R N A
WITH N-BROMOSUCCINIMIDE
(NBSI) AT
P H 7 AND 9
Nucleotid* ratios Adenylic acid
TMV-RNA After b r o m i n a t i o n at p H 7, nucleotide: N B S I 5 : I After b r o m i n a t i o n at p H 9, nucleotide: N B S I io: i nucleotide: N B S I 5 : i
Guanylic a c i d
Uridylic acid
Cytidylic acid
I
0.87
o.91
0.66
i
0.76
0.88
0.56
i i
0.79 0.65
0.90 o.87
0.62 0.56
reaction. Each set of figures represents the average of three separate determinations and the maximum error associated with each figure is 2 %. In the reaction with N-bromosuccinimide at both pH 7 and 9, guanine and cytosine represent the most reactive sites. At pH 7 the proportion of each decreases by about 15%, whereas, at a similar level of N-bromosuccinimide at pH 9, the guanine content decreases 25 % and the cytosine about 15 %. The change in uridylic acid content is significant, but only just detectable by this method. The brominated infectious TMV-RNA from which mutants were isolated was obtained by reaction with N-bromosuccinimide at pH 7, so it appears that the mutagenic event might have been either due to a change in cytosine or guanine. Reaction with bromine
Using the same conditions as for the reaction with N-bromosuccinimide the rate of reaction of the separate nucleotides with bromine was similarly studied. The 100
gO
.= o
,....(
8(3
~°o
2'0
4'0
Time (rnin)
do
do
Fig. 3. Reaction of guanylic acid w i t h b r o m i n e at p H 7 ( O - - O ) and 9 ( 0 - - - 0 ) . Nucleotide concentration, , ~ i o /*moles/ml. B r o m i n e concentration, ~ 5 /,moles/ml. 5 ° ;t of each mixed in presence of :o 2 of buffer.
reaction with the pyrimidine nucleotides was almost instantaneous at both pH 7 and 9. Bromination of cytidylic acid resulted in the formation of the new ultraviolet absorption peak at 237 m/z in each case. The reaction of guanylic acid with bromine was considerably faster at pH 9 than at pH 7 (Fig. 3). Once again adenylic acid was completely stable to bromination. Biochim. Biophys. Acta, 72 (1963) 217-229
222
K.W.
BRAMMER
The change in the nucleotide composition of RNA after treatment with bromine (I mole Brz:io moles nucleotide) was studied at pH 7 and 9, aliquots of the brominated RNA being taken after 15 min and 2 h. The results obtained are shown in Table II. These figures indicate that at pH 7 and using a low level of bromine, only cytosine in the RNA is attacked. At pH 9 the specificity is quite different and guanine
TABLE REACTION OF T M V - R N A
II
WITH BROMINE AT P H 7 AND 9
Nuvleotide ratios
TMV-RNA B r o m i n a t i o n a t p H 7, nucleotide-Br v io:i 15 r a i n 2 h B r o m i n a t i o n a t p H 9, nucleotide--Br v io:i 15 m i n 2 h
Adenylic acid
Guanylic acid
Uridylic acid
Cytidylic acid
i
0.88
0.92
o.66
i I
0.88 0.87
0.92 o.91
0.56 0.53
i i
o.86 o.81
o.89 0.92
0.66 0.66
seems the only base brominated. This change in specificity is much more dramatic than was obtained using N-bromosuccinimide, but with that reagent too, the bromination of guanine appeared to be favored at pH 9.
Bromination products of cytidylic and guanylic acids The foregoing experiments indicated that the guanine and cytosine residues in RNA were the favored sites of bromination using fairly low levels of brominating agent at either pH 7 or 9. An attempt was made to identify the products of bromination of cytidylic and guanylic acids under these conditions. Cytidylic acid: The bromination of uridylic acid, uridine, and other uracil derivatives has been studied in detail by several workers 1°-1~, and it was established that at pH 7 one mole equivalent of bromine was absorbed and caused complete loss of the characteristic uracil ultraviolet absorption peak. This was due to saturation of the 5,6 ethylenic linkage in uracil by the formation of a 5-bromo-6-hydroxyhydrouracil d'erivative 11,12. The latter compound was unstable and either heating or exposure to acidic pH resulted in the reappearance of an ultraviolet absorption spectrum due to elimination of water from the 5,6 position forming a 5-bromouracil derivative. This compound could then react with another mole equivalent of bromine to form a 5,5dibromo-6-hydroxyhydrouracil derivative. The present work has shown that the bromination of cytidylic acid proceeds in a similar manner. The first bromination product of cytidylic acid using bromine or N-bromosuccinimide at either pH 7 or 9 had an absorption maximum at 237 m#. This was the s a m e ~.max as found for 5,6-dihydrocytosine derivatives is and appeared to be B i o c h i m . B i o p h y s . A c t a , 72 (1963) 2 1 7 - 2 2 9
BROMINATION AND IODINATION OF TMV-RNA
22 3
characteristic for derivatives of cytosine in which the 5,6 ethylenic linkage was saturated. Provided that excess brominating agent was absent, the action of heat or acid on this brominated derivative resulted in the loss of the ultraviolet absorption peak at 2,37 m/z and the formation of a new peak at 287 m/~. Further bromination resulted irr the disappearance of the latter peak and a compound having an absorption peak at 237 m# was again produced. The same sequence of reactions as proposed for uracil derivatives by WANGTM and MOORE AND ANDERSONn apparently took place with cytidylic acid. That is, NHz
NH,
I
I
/tiC\ N C--H
I
I
C--H
I
R
>
•
NH,
I
, / C \ --H N C~,B r
Br, H,O
NH,
Heat
|
/C~IH
°rH+
I
÷
I
I
.C
C--H
I
R
I
/tiC\ N C--Br
R
//C\ Br N C~B r
Br, H20
>
I
I
H
]
R
Cytidylic acid, 5-Bromo-6-hydroxy- 5-Bromocytfdylic acid, 5,5-Dibromo-6-hydroxy~.max (H,O) ~= 287 m/~. hydrocytidylic acid, ~max (HzO) = 270 m#. hydrocytidylic acid, ~max (H,O) 237 rn/~. )lmax (H20) = 237 m/~.
Saturation of the 5,6 ethylenic linkage in cytosine derivatives still leaves the
J
f
conjugated chromophore --C ---- N -- C = O to which the absorption peak at 237 m/z is attributed. The structure of the 5,6-saturated uracil derivatives in the favored keto form does not have this chromophore, nor do they have an absorption peak above 21o m/z. 5-Bromocytidylic acid was readily separable from cytidylic acid on electrophoresis at pH 3.5 under the standard conditions for nucleotide separation. The mobility of 5-bromocytidylic acid was o.81 relative to that of uridylic acid (very similar to guanylic acid), whereas cytidylic acid had a mobility of 0.20 relative to uridylic acid. When bromination mixtures of cytidylic acid with either bromine or N-bromosuccinimide were separated directly by electrophoresis at pH 3.5, cytidylic acid and 5-bromocytidylic acid were the only compounds detectable by absorption of ultraviolet light. This indicated that 5-bromo-6-hydroxyhydrocytidylic acid was rapidly converted to 5-bromocytidylic acid at pH 3.5. It was shown that this conversion took place within 5 h at room temperature at any pH less than 6. The following were the ultraviolet absorption characteristics for 5-bromocytidylic acid, the emax being the absorbancy per mole of phosphorus: in water, emax 6800; ~max 287 m/~; in o.oi N HC1, emax 9000; ~.max 298 m/~. When 5-bromo-6-hydroxyhydrocytidylic acid was set aside at room temperature and alkaline p H (pH 8-I3), the absorption peak at 237 m/z disappeared with the formation of a new peak at about 29o mp in water. The reaction was very slow at pH 8 but was complete within 2 h at pH 13. The following properties established that it was not 5-bromocytidylic acid: in water, emax 7500; ~max 287 m/z; in o.oi N HC1, emax 9300; Amax 3Ol mp; in o.oi N NaOH, emax 7oo0; Amax 316 rap. The mobility of the compound on electrophoresis at pH 3.5 was 0.44 compared Biochim. Biophys. Acta, 72 (1963) 217-229
224
K. W. BRAMMER
with uridylic acid. This was similar to the mobility of adenylic acid and considerably less than that of 5-bromocytidylic acid (o.81). Both cytidylic and 5-bromocytidylic acids have a similar ~max in water and in alkali, whereas there was a difference of about 30 m # in this case. FUKUHARA AND VISSER14 probably obtained the nucleoside derivative of this same compound by passing a solution of 5-bromo-6-hydroxyhydrocytidine through a column of a basic resin in the O H - form. They recorded emax 7837, ~tmax 292 m/~, for the compound at p H 7 and presented evidence suggesting that it was 5-hydroxycytidine. These experiments concerning the stability of the first bromination product of cytidylic acid indicated that it was necessary to incubate RNA after bromination at a mildly acidic p H to ensure that bromination of cytosine resulted in formation of the relatively stable 5-bromocytosine. Guanylic acid: The bromination of guanylic acid was not studied in such detail, but a derivative was obtained which absorbed ultraviolet light on treatment of guanylic acid with bromine at p H 7. The spectrum in acid had a slight hump at about 280 in/z, but not as pronounced as that of guanylic acid itself. In alkali the spectra of both guanylic acid and the brominated derivative had a single peak, but at a different wavelength. A comparison of the ultraviolet absorption characteristics of these compounds is shown in Table I I I . TABLE
III
C O M P A R I S O N OF U L T R A V I O L E T A B S O R P T I O N P R O P E R T I E S OF G U A N Y L I C ACID A N D B R O M I N A T E D
Guanyl i c acid Medium
o.oI N HCI o.oi N NaOH
GUANYLIC ACID
B r o m i n a t e d guanylic acid . . . . . . . . . . .
*ma*
~max (ma)
e,n~,
~. . . . {into
12 2oo I I IOO
257 258-263
9ooo 74oo
26o 27o
The distinct similarities in ultraviolet absorption spectra of the brominated derivative and parent compound suggested that they were still closely related structurally. I t was more likely that the bromination was a substitution reaction rather than an addition. It appeared probable that this was the 8-bromoguanine derivative as identified in acid hydrolysates of brominated RNA by KANNGIESSER3. The electrophoretic mobility of the brominated compound at p H 3.5 was slightly less (0.76) than that of guanylic acid (o.81). The same compound was apparently produced when guanylic acid was treated with less than I mole equivalent of N-bromosuccinimide at p H 7, but further bromination to non-ultraviolet-absorbing products occurred when excess of the reagent was present. The bromination of guanylic acid at p H 9 using either bromine or Nbromosuccinimide appeared to follow a different course since in neither case was a compound formed which absorbed ultraviolet light.
Iodination with N-iodosuccinimide The relative rates of iodination of the separate nucleotides at p H 7 and o ° were determined b y comparing the rates of decrease in ultraviolet absorption as previously Biochira. B i o p h y s . A c t a , 72 (1963) 2 1 7 - 2 2 9
BROMINATION AND IODINATION OF T M V - R N A
225
described for the bromination experiments. Reaction was detected only with cytidylic and uridylic acids (Fig. 4). The iodination of cytidylic acid was a much faster reaction than the iodination of uridylic acid under the same conditions. The extent of reaction was limited b y the decomposition of N-iodosuccinimide in aqueous solution which liberated free iodine.
100,
90
@
8o
o x•
70
E •~
60
"6
5%
io
go
15o
1~o
Time (min)
Fig. ¢. Reaction of nucleotides with N-iodosuccinimide at pH 7.0. (Conditions as in Fig. i). AMP and GMP ( A - - A ) , CMP (0---0) and UMP ( O - - O ) .
When a similar series of reactions was conducted at p H 9, uridylic acid was the only nucleotide in which a decrease in ultraviolet absorption was detected and in this case the decrease was only slight (about 5 % in I h). One of the factors influencing this diminished reactivity of N-iodosuccinimide was probably the faster decomposition of the reagent in aqueous solution at p H 9 compared with p H 7. Since cytidylic acid was clearly the most rapidly iodinated nucleotide at p H 7, a more detailed study of this reaction was made. Cytidylic acid was treated with 2 mole equivalents of N-iodosuccinimide in lO -3 M E D T A (pH 7) at o ° for I h. Iodine and other ether-soluble products were removed b y four ether extractions and the aqueous phase was concentrated and investigated by paper electrophoresis at p H 3.5 for 3 h. Markers of the four nucleotides were also applied and separated. The iodination mixture contained two components which absorbed ultraviolet light, one having a low mobility which was shown to be unreacted cytidylic acid, while the other had a mobility of o.81 compared to uridylic acid. This was the same mobility as guanylic acid and also the same as that found previously for 5-bromocytidylic acid. This zone was eluted in water and was shown to have the following ultraviolet absorption characteristics, based on phosphorus analysis: in water, emax 5800; ~max 292 m/~; in o.oi N HC1, emax 7700; ;tmax 307 In#. A comparison of these figures with the corresponding values determined for 5bromocytidylic acid shows that the emax values of the iodinated derivative are slightly lower and that the absorption maxima occur at slightly higher wavelengths than those of the brominated derivative. The behavior of the ultraviolet absorption spectra Biochim. Biophys. Aaa, 72 (1963) 217-229
226
K. W. BRAMMER
on acidification is similar with both compounds. These similarities of spectra, together with the fact that both compounds exhibit the same mobility on electrophoresis at p H 3.5 (indicative of the same p K value) led to the conclusion that the iodinated derivative was 5-iodocytidylic acid. Infectious TMV-RNA was treated with inactivating levels of N-E131I]iodosuccinimide at p H 7 for I h and after 6 precipitations of the iodinated RNA with ethanol the number of atoms of iodine bound per 6400 nucleotides was calculated for the various samples. Aliquots of these same samples were reconstituted with TMVprotein and assayed for infectivity. The results of several such experiments (Table IV) indicated that the binding of about 4 atoms of iodine per 6400 nucleotides in the total RNA population was necessary to cause the infectivity of the RNA to be reduced to 37% of the control RNA (i.e., the inactivation produced by one lethal hit). TABLE I N A C T I V A T I O N OF I N F E C T I O U S T M V - R N A
Atoms of I #er 6400 nucleolide8 o 0.5 1.2 2.3 2.5 3.5 4.3 5.5 12. 7
IV CAUSED BY B I N D I N G OF I O D I N E
Infeaivity (% of control) IOO 9o ~IOO 90 65 5° 4° 4 0. 5
For investigation of the actual iodination site in the RNA a sample of noninfectious RNA was iodinated with a higher level (approx. I mole reagent: 2 moles nucleotide) of the N-E131I~iodosuccinimide and then precipitated with ethanol until the ratio of counts/min to absorbancy was constant. In this preparation about 84 atoms of iodine were bound per 6400 nucleotides. The iodinated RNA was degraded and the products separated on paper in two ways. (a) By alkaline hydrolysis to the nucleotides and separation of the nucleotide mixture b y the method combining paper chromatography and p H 3.5 electrophoresis as described previously. (b) By total digestion to nucleosides using the mixture of phosphodiesterases and phosphomonoesterases present in crude snake venom. The nucleosides were then separated on paper using the 2-dimensional system of FELIX et al. ~. Both papers were radioautographed and in each case most of the radioactivity (but no ultraviolet-absorbing material) remained at the origin indicating that considerable decomposition of the iodinated derivative had occurred during the digestion procedure. The only other radioactive area present on the nucleotide paper was associated with the guanylic acid zone. This was the expected position for 5-iodocytidylic acid as determined previously. The nucleoside paper also had only one radioactive area besides the origin and Biochim. Biophys. Acta, 72 ( 1 9 6 3 ) 2 1 7 - 2 2 9
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this was located in a position between the purine and pyrimidine nucleosides. A marker of 5-iodocytidine chromatographed in the same 2-dimensional system had a similar position relative to the normal nucleosides. Difficulty was experienced in identifying compounds by their RF values in this system since mobilities in both solvents were sensitive to temperature change and the degree of saturation of the atmosphere in the chromatography cabinet. The evidence of this experiment, together with that from the reaction rate studies on the separate nucleotides, suggested strongly that cytosine was the RNA component most readily iodinated by N-iodosuccinimide at pH 7. Insufficient counts remained on the chromatograms to permit further establishment of the identity of the iodinated compound.
Iodination using iodine in K I solution No change in the ultraviolet absorption spectra of the nucleotides was detected on treatment with iodine in potassium iodide solution at o ° at either pH 7 or 9. A slow decrease in the ultraviolet absorption maximum was detected, however, upon iodination of uridylic acid at 37 ° in carbonate-bicarbonate buffer (lO -2 M) at pH 8-1o. This decrease was more rapid at pH IO than 8 but in no case was the final absorption less than 80% of the original, although free iodine was still present. Iodination of infectious TMV-RNA was attempted in o.oi-o.I M carbonatebicarbonate buffers varying in pH from 8 to 9.5 at 37 °, but in no case was there any inactivation beyond that of the control RNA. When experiments were carried out under the same conditions with 181Ino evidence of the binding of iodine was obtained. In many of these experiments considerable inactivation of infectious TMV-RNA was brought about by the conditions of reaction so it was concluded that iodine itself was not a sufficiently reactive iodinating agent to be of any value in these studies.
The relative efficiency of the halogenating reagents for reaction with TMV-RNA The inactivation of infectious TMV-RNA using different levels of halogenation reagent was studied. All experiments were performed at o ° for I h after which the modified RNA was twice precipitated with ethanol, then reconstituted with TMVprotein and assayed for infectivity. The' reaction with N-bromosuccinimide and N2
"E 100
8 8O
:>_- 60 .~ 40 ._=o, .£_ 20 E ,,. o
100 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 700 8 0 0 9 0 0 Moles r e a g e n t added per mole RNA
1000
Fig. 5- Inactivation of TMV-RNA by halogenation reagents. Br s at pH 7 ( / k - - A ) , Bra at p H 9 ( A - - A ) , N-bromosuccinimide a t pH 7 (O--O) and N-iodosuccinimide at p H 7 ( G - - O ) . Biochim. Biophys. Acta, 72 (1963) 2x7-220
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K. W. BRAMMER
iodosuccinimide were studied at pH 7 and the reaction with bromine at pH 7 and 9. The relative efficiency of bromine and N-bromosuccinimide for the inactivation of TMV-RNA at pH 7 are quite similar. Bromine at pH 9 is significantly less efficient. The iodination using N-iodosuccinimide at pH 7 is a much less efficient inactivating reaction requiring approximately a Io-fold excess of N-iodosuccinimide compared with the levels of brominating agent needed to produce similar inactivation under the same conditions (Fig. 5). DISCUSSION
From the point of view of finding reactions which chemically modify TMV-RNA, preferably by reacting specifically with only one of the bases, both bromination and iodination under the correct conditions are of interest. The only halogenation reagent wich has been established as definitely mutagenic towards TMV is N-bromosuccinimide acting at pH 7. The present work shows that this leads most probably to bromination of guanine or cytosine (or both). Since it is not known whether only one, both or neither of these events causes the mutations, correlation of the reaction with the RNA to the change induced in the mutant protein is made difficult. It is of interest to note, however, that the changes in amino acid composition of mutant proteins induced by this reaction show marked similarities to those in some mutants obtained by the action of nitrous acid on infectious TMVRNA 4. Bromine itself is shown to produce more selective bromination than N-bromosuccinimide. Low levels of reaction with RNA at pH 7 cause almost exclusive bromination of cytosine, whereas, at pH 9 tt:e reaction is similarly specific for the bromination of guanine. After bromination at pH 7 it is desirable to expose the modified RNA to mildly acidic conditions before reconstitution to favor the formation of the relatively stable 5-bromocytosine rather than other products. No informatizn per~aining to the nature of the products obtained on reaction of guanine derivative; with bromine at pH 9 is available at present. Bromination of guanine compounds at pH 7 probably results in formation of the 8-bromoguanine derivative. It has not yet been established whether the reactions of bromine with TMV-RN~', at pH 7 and/or 9 lead to mutation of the virus*. If only one of these reactions leads to mutation, it might be possible to determine the nature of the mutagenic change which occurs on reaction with N-bromosuccinimide. In any case it would be of interest to make a comparison of the amino acid composition of mutant proteins produced by different bromination reactions. The reaction of TMV-RNA with N-iodosuccinimide at pH 7 favors the iodination of cytosine. This reaction does not appear to be mutagenic. It is possible that the iodine is too large an atom to cause a mutagenic change and is only inactivating. The data in Table IV indicates that the binding of about 4 atoms of iodine per 64oo nucleotides is necessary to produce the inactivation of one lethal hit. Since the RNA preparations used consist of only 50-80% of full-length, infectious molecules, the
* A conclusive s t u d y of the action on TMV of m u t a g e n s of low efficiency is at p r e s e n t n o t possible in our laboratory. This is due to the u n u s u a l l y high frequency of w h a t a p p e a r to be s p o n t a n e o u s m u t a t i o n s observed during the p a s t year in our greenhouse.
Biochim. Biophys. Acta, 72 (1963) 217-220
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TMV-KNA
229
number of iodinations which cause loss of infectivity is 2-3 per infective RNA molecule. This is similar to the number of alkylations necessary to produce the same inactivation of TMV-RNA 1. ACKNOWLEDGEMENTS
The author expresses his thanks to Dr. H. FRAENKEL-CONRATfor his advice and helpful discussion during the course of this work and for making the laboratory facilities available, and to Mrs. B. SINGER for discussion and advice on practical techniques. This investigation was supported by a grant from the National Science Foundation. REFERENCES I H. FRAENKEL-CONRAT,Biochim. Biophys. Acta, 49 (1961) 169. i A. S. JONES AND D. L. WOODHOUSE,Nature, 183 (1959) 16o 3. 8 W. KANNGIESSER, Z. Physiol. Chem., 316 (1959) 146. 4 A . TSUGITA AND H. FRAENKEL-CONRAT, J. Mol. Biol., 4 (1962) 736 A. TSUGITA, J. Mol. Biol., 5 (1962) 284. t H. FRAENKEL-CONRAT,B. SINGER AND A. TSUGITA, Virology, 14 (1961) 54" 7 A. GIERER AND G. SCHRAMM, Z. Naturforsch., I I b (1956) 138. s H. FRAENKEL~CONRAT AND B. SINGER, Biochim. Biophys. Aaa, 33 (1959) 359. o R. MAEKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 552. to W . E. COHN, Biochem. J., 64 (1956) 28. 11 A. M. MOORE AND S. M. ANDERSON, Can. J. Chem., 37 (1959) 590. ts S. Y. WANG, Nature, 18o (1957) 91. is D. SHUGAR, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol., 3 196o, p. 53. 14 T. K. FUKUHARA AND D. W. VISSER, Biochemistry, I (1962) 563 . 15 F. FELIX, J. L. POTTER AND M. LASKOWSKI, f . Biol. Chem., 235 (196o) II5O.
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