The oxygen-dependent reaction of hydroxylamine with nucleotides and DNA

I7

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95408

T H E O X Y G E N - D E P E N D E N T REACTION OF H Y D R O X Y L A M I N E

WITH

NUCLEOTIDES AND DNA

E R N S T F R E E S E , E L I S A B E T H BAUTZ F R E E S E AND STUART GRAHAM

Laboratory o/Molecular Biology, National Institute o/Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Md. (U.S.A.) (Received October 4th, 1965)

SUMMARY

Hydroxylamine reacts, at low concentrations (IO-~ M), with DNA, as exhibited by the initial increase (melting) and later decline of the extinction at 260 m# which can be observed at temperatures far below the melting temperature of DNA; the reaction rate increases with the pH. Under the same conditions several nucleotides show a reaction which has not been observed at high hydroxylamine concentrations. All these reactions are oxygen dependent, and they are quenched by catalase, peroxidase and Tris, but most effectively by pyrophosphate. This indirect effect of hydroxylamine is contrasted to the direct effect of high hydroxylamine concentrations on cytosine and uracil and is explained by the production of peroxides and free radicals.

INTRODUCTION

Hydroxylamine and its derivatives, containing an unesterified NOH group, exert two biological effects on DNA: a predominantly mutagenic and a predominantly inactivating effect 1. Whereas the mutagenic effect increases linearly with the hydroxylamine concentration, is strongest at slightly acidic pH and is 02 independent, the inactivating effect is strongest at some intermediate hydroxylamine concentrations (I0 -~ M), at alkaline pH and is found only in the presence of 02 (ref. 2). This paper investigates the O~-dependent effect of hydroxylamine on nucleotides and DNA, as revealed by spectrophotometric observations under different reaction conditions. MATERIALS AND METHODS

Escherichia coli DNA, highly polymerized, was obtained from General Biochemicals Corp., Chagrin Falls, Ohio; poly d(A-T) was kindly given to us by Dr. Abbreviation: poly d(A-T), alternating regular sequence dA-dT.

Biochim. Biophys. Acta, 123 (1966) 17-25

I8

E. FREESE, E. B. FREESE, S. GRAHAM

A. Kornberg, Stanford University; NH~OH-HC1 was from Fluka Chemische Fabrik, Buchs SG, Switzerland; deoxynucleotides, from Sigma Chemical Co., St. Louis, Mo. Saline-citrate: o.I M NaC1 plus o.oi M sodium citrate. Reactions at p H 9 were run in sodium borate buffer (o.I M), except when specially stated. Spectra were read in the Cary recording spectrophotometer (Applied Physics Corp., Monrovia, Calif.) and kinetic as well as melting curves were determined by the Gilford recording spectrophotometer (Oberlin, Ohio).

RESULTS

Reactions with nucleotides The reaction of DNA and RNA bases had been measured only at high hydroxylamine concentrations (I M) at which a strong mutagenic effect predominates. Of all DNA or RNA bases, only C (refs. 3, 4) and U (ref. 5) exhibited reactions. With increasing pH the reaction rates decreased for C and increased for U (refs. 5, 6). a. In order to determine which bases react with hydroxylamine under conditions of a strong inactivating effect on DNA, we observed the change in ultraviolet absorption of different nucleotides in I o - ~ M h y d r o x y l a m i n e and o.I M sodium borate buffer of p H 8 or 9 at 5 °o or 7 o°. In addition to UMP and dCMP a reaction was also observed for dTMP and dGMP as shown in Fig. I. The change in absorbance could not be explained merely b y a pH change, because the p H at the end of the experiment was the same as at the beginning. Furthermore, an A decrease was also observed when the reaction mixture was acidified (to 0.5 M HC1) after different reaction times. Without hydroxylamine no A change was registered for any nucleotide. 1.0'

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Biochim.

Biophys.

Acta,

123 (1966) 17-25

19

OXYGEN EFFECT OF HYDROXYLAMINE ON D N A

Hydroxylamine alone also produced some ultraviolet-absorbing compound whose absorption maximum was below 21o mF. This material caused the increase in ab-

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Fig. 2. R a t e s of nucleotide reactions a t different h y d r o x y l a m i n e concentrations. The absorbance w a s recorded at 26o In# for d A M P (no reaction) and at (a) 272 m/~ for dCMP, (b) 253 m # for dGMP, (c) 267 m # for dTMP, (d) 262 mff for UMP. The r a t e s are defined as dA/dAt, dt where A 1 is the initial a b s o r b a n c e at zero t i m e (time in hours). 0 , o.I M s o d i u m b o r a t e (pH 9.o) at 50°; O, s a m e plus o.05 M NaaP20 ~ (pH 9.0). T h e a r r o w s indicate r a t e s t h a t are smaller t h a n the value m a r k e d b y a dot.

Biochim. Biophys. Acta, 123 (1966) 17-25

20

E. FREESE, E. B. YR.EESE, S. GRAHAM

sorbance observed below 24o m/~ in Fig. I. dAMP exhibited only a 2.5~o decrease in ultraviolet absorption in 5 days. b. In order to establish the unusual concentration dependence of the above reaction we recorded continuously (for about 20 h) the A decrease of the nucleotides at different hydroxylamine concentrations. Cuvettes containing hydroxylamine and sodium borate buffer (final conch, o.I M, pH 9) were equilibrated at 5°0 for 5-1o min before I/IO volume of a IO-~ M solution of the nucleotide was added. The cuvettes were stoppered and the decrease of absorbance was recorded at the wavelength of the respective absorption maxima. The reaction rates are plotted against the hydroxylamine concentration in Fig. 2. At hydroxylamine concentrations ~> o.I M the rates of both dCMP and UMP increased linearly with the hydroxylamine concentration, as expected for a direct effect of hydroxylamine on the bases, dTMP and dGMP, however, exhibited much higher reaction rates at low than at high hydroxylamine concentrations. This abnormal concentration dependence is similar to that observed for the O~-dependent inactivating effect of hydroxylamine on transforming DNA1, 2. The indirect effect of hydroxylamine seems to affect also UMP, which still exhibited a significant reaction at low hydroxylamine concentrations (see Fig. 2). c. The reactions of dTMP, dGMP, and UMP, observed at low hydroxylamine concentrations, were eliminated when the mixture contained 0.05 M Na 4 P207. In contrast, the reactions of dCMP or UMP at high hydroxylamine concentrations were not influenced by pyrophosphate (see Fig. 2).

Melting o/ D N A When DNA is exposed to hydroxylamine its melting temperature gradually decreases. a. This has been shown for I M hydroxylamine at pH 7.5 by TROLL, BELMAN AND LEVINE7 and for o.oi M and o.I M hydroxylamine at pH 7.0 by BENDICH et al. s. In both cases the decrease in melting temperature has been explained by the direct reaction of hydroxylamine with cytosine alone, which reduced or eliminated the hydrogen bonding of the GC pair. b. In order to measure the effect of hydroxylamine on DNA under conditions under which the inactivating effect was strong while dCMP reacted only little, we treated DNA (E. coli) with lO -3 M hydroxylamine at pH 9. After reaction for 0.5 or I h at 65 ° an equal volume of ice-cold o.I M pyrophosphate (pH 7) was added and the mixture was dialyzed extensively against ice-cold saline-citrate. A control sample was treated for I h under the same conditions, but without hydroxylamine. Fig. 3 shows that the treated DNA exhibited not only a greatly reduced melting temperature but also a reduced total increase in extinction, indicating that even at low temperatures a portion of the DNA bases was no longer able to retain the double helical configuration. c. The increase of the absorption could also be observed during the reaction of hydroxylamine with DNA, as is shown in Fig. 4 a. After an initial lag the absorbance at 26o m# began to rise at a rate that increased with the temperature. At each temperature the absorption reached a maximum and then declined. The curves may be roughly characterized by three parameters, the maximum rate (at the inflexion Biochim. Biophys. Acta, 123 (1966) 17-25

OXYGEN EFFECT OF HYD ROXYLAM I NE O N L)INA

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Biochim. Biophys. Acta, 123 (1966) 17-25

22

E. FREESE, E. B. FREESE, S. GRAHAM

point) of A increase, a lag period, which m a y be defined as the intersection between the tangent to the inflexion point and the horizontal line through the initial absorbance, and the rate of decrease following the attainment of a maximal A. The m a x i m u m rates are plotted against I/T in Fig. 5.

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Fig. 5. M a x i m a l r a t e s of A260m F increase (melting) of D N A in h y d r o x y l a m i n e . E. coli D N A in zo -2 M h y d r o x y l a m i n e : × , o.o5 M Na2CO 8 (pH 9); O , 0.05 M Na2CO 3 (pH 8); g , o.I M s o d i u m b o r a t e (pH 9); /x, o.i M s o d i u m b o r a t e + o . o 5 M Na4P207 (pH 9); ~ , o.I M Tris (pH 9); @, o.I M Tris (pH 8); A, c o n t r o l of o.I M s o d i u m b o r a t e (pH 9) w i t h o u t h y d r o x y l a m i n e . E. coli D N A in o. 9 M h y d r o x y l a m i n e : A , o.i M s o d i u m b o r a t e (pH 9); [], o.05 M Na2CO 3 (pH 9). E], poly d ( A - T ) in lO -2 M h y d r o x y l a m i n e a n d o.I M Tris (pH 8). L a g t i m e s were e.g. for E. coli D N A , in io -2 M h y d r o x y l a m i n e plus b o r a t e (pH 9): 44 m i n a t 56°, 20 m i n a t 65 °, a n d 5 m i n a t 75 °. Fig. 6. E f f e c t of r a p i d cooling on t h e e x t i n c t i o n of E. coli D N A t r e a t e d b y io 2 M h y d r o x y l a m i n e (pH 9). D N A w a s t r e a t e d , in s t o p p e r e d c u v e t t e s , for d i f f e r e n t t i m e s b y h y d r o x y l a m i n e . T h e c u v e t t e s were s u d d e n l y chilled b y ice-water, w a r m e d u p to r o o m t e m p e r a t u r e a n d t h e A260 m# was again measured. T h e A260m# of a control mixture, containing hydroxylamine but no DNA, continuously increased but at a much lower rate (0.35 A units in IO h). If DNA, however, would catalyze the production of such ultraviolet-absorbing products derived from hydroxylamine, their absorption could conceivably greatly contribute to the observed A increase. In order to estimate this effect, the hydroxylamine-DNA solution, after reacting for different times, was exposed to 0.2 M N a O H which denatured the DNA. At the time of m a x i m u m A, the A increase thus obtained was only 5o % of that at zero time and after 3 h at p H 9 it was only 30 %. Since denatured DNA also shows such an A increase when it is taken from p H 8 or 9 into 0.2 M NaOH, it is clear that the major portion of the observed A increase was caused b y the denaturation of DNA itself. d. In order to show that the reaction of DNA observed here corresponded to the O2-dependent DNA inactivating effect, we performed the following controls: (I) When 02 was replaced by N 2 (by evaporation and flushing with N2) much less reaction was observed (Fig. 4a). (2) Catalase, peroxidase, and E D T A (lO -4 M) also inhibited the reaction, but the most efficient inhibitor was 0.o5 pyrophosphate (Fig. 4 b) as had been observed already for the inactivating effect of hydroxylamine on DNA 2. o. I M Tris also partially reduced the melting rate in lO -2 M hydroxylamine (see Figs. 4b and 5)- Quite similarly, Tris reduced the rate of DNA inactivation by lO -2 M hydroxylamine and p H values of 7.5 or 9. Biochim. Biophys. Ac/a, 123 (1966) 17 25

OXYGEN EFFECT OF HYDROXYLAMINE ON

DNA

23

(3) In o. 9 M hydroxylamine the absorption increased only very slowly (see Fig. 4c); i.e. the reaction of DNA was much stronger at low than at high hydroxylamine concentrations. e. In IO-z M hydroxylamine, poly d(A-T) also showed an A increase below its normal melting temperature of 65 ° . The experiments, using up the small supply of d(A-T), were unfortunately performed in o.I M Tris (pH 8) before we realized the quenching effect of Tris. Nevertheless, the observed rates of A increase compare well with those found for E. coli DNA in o.I M Tris (see Fig. 5). f. Finally, we have shown that the reaction of DNA with hydroxylamine did not irreversibly destroy the ability to form an ordered structure for all bases which had opened out of the helical array. To this end the stoppered cuvettes were suddenly immersed in ice-water at different times of the reaction and the absorption was measured again. Fig. 6 shows that a small part of the increase in absorption was reversible on cooling.

DISCUSSION

Parallel to the two types of biological reactions on transforming DNA ~,2, hydroxylamine exhibits two distinct chemical reactions with nucleotides and DNA.

Reaction with nucleotides a. At high hydroxylamine concentrations (I M) hydroxylamine directly reacts with C or its nucleotides at neutral or acidic pH and with U or its nucleotides at basic pH; the reaction mechanism is essentially known in both cases3-e, 9-11. It is this reaction with cytosine which induces mutations in phages3,4,12,13 and transforming DNA, the bases in denatured DNA being much more sensitive than in undenatured DNAL14,15. A change in the base-pairing properties has also been observed for hydroxylamine-treated poly-C that was copied by RNA polymerase (refs. 16, 17). b. At low hydroxylamine concentrations (I0-2 M) we have spectrophotometrically observed a new reaction with dGMP, dTMP, and UMP, which is apparently 02 dependent and whose rate increases with the pH. This reaction is quenched at high hydroxylamine concentrations (I M) or in the presence of pyrophosphate. These findings indicate that the reaction involves the formation of peroxides and radicals as has been discussed in detail previously ~. In addition nitroxyl (HNO or :NOH) is probably formed, which reacts further to produce nitrite, hyponitrite, etc. Whereas it is clear that the radicals formed here (such as OH) can attack certain nucleotides, it is not known whether some nucleotides can also react with nitroxyl. All other reaction products of hydroxylamine oxidation seem to be excluded as the reactive species, as has been discussed previously z. A chromatographic analysis indicated that most reaction products are the same as those obtained after treatment by H202 (FREESE, MELZER AND RHAESE, unpublished results). The effect of low concentrations of hydroxylamine m a y therefore be the same as that of H202, both being caused by OH radicals. That analysis has furthermore shown that not only the above nucleotides but also dAMP react Biochim. Biophys. Acta, i 23 (1966) 17-25

24

E. FREESE, E. B. FREESE, S. GRAHAM

by having the base cleaved away from the deoxyribose phosphate. For dAMP this reaction would be difficult to observe spectrophotometrically, because A and dAMP have nearly the same absorbance coefficient. KOCHETKOV, BUDOWSKY AND SHIBAEVA18 spectrophotometrically observed that propiohydroxamic acid (C2HsCNHOH) reacts with purine and pyrimidine nucleosides having enolizable keto-groups, including guanosine, thymidine, and uridine. The authors assumed a direct reaction of the hydroxamate with the bases. The 02 independence of this reaction has not been established. The reaction with D N A

The melting temperature of DNA decreases when it is exposed to hydroxylamine. a. At high hydroxylamine concentrations and neutral pH, the effect can be attributed to the direct reaction of hydroxylamine with cytosine, opening the GC base pairs onlyT,s. b. At low hydroxylamine concentrations (lO -2 M) and pH 9, however, the reduction of the melting temperature is caused by a different reaction which depends on 03. This reaction occurs much faster at lO -2 M hydroxylamine than at I M hydroxylamine and it is suppressed by O3 deficiency, catalase, peroxidase, or 0.05 M pyrophosphate (see Fig. 4). The 02-dependent effect corresponds to the predominantly inactivating effect observed for transforming DNA. It apparently depends on the formation of peroxides and radicals. Eventually, complete melting seems to be obtained in lO -2 M hydroxylamine, even at 45 °, which is much below the melting temperature of the E. coli DNA used (9o°) and also below the melting temperature of poly d(A-T)(65°). Following the initial increase, the absorbance at 260 m# of a DNA solution, in lO -3 M hydroxylamine at pH 9, decreases (see Fig. 4a). This decrease presumably reflects both the liberation of bases from now denatured DNA and the further reaction of these bases. The A260m/~ increase and decrease reveals a massive reaction of the DNA nucleotides. DNA usually cannot duplicate across a single gap or a drastically altered base, unless the alteration is repaired. The inactivation of transforming DNA by low concentrations of hydroxylamine can therefore be explained by the removal or reaction of these bases alone. BEI~DICH et al. s observed that hydroxylamine treatment did not only reduce the viscosity of DNA (which could be explained by melting alone) but also the sedimentation constant. Similarly, SCHWEITZ AND LUZZAT119have observed a considerable decrease of the sedimentation constant of DNA after treatment with H202. In neither case is it known whether the radicals produced can directly break the DNA backbone or whether they rather act indirectly by removing DNA bases. Peroxides are continuously produced by the reaction of hydroxylamine with O, and radicals are continuously formed by both this reaction and the subsequent reaction of peroxide with hydroxylamine. The latter reaction is actually most effective because the combination of H~O2 and hydroxylamine reacts faster with DNA than either agent alone 2. The continuous production of radicals seems ideal for the controlled chemical inactivation of nucleic acids, which is accompanied by only a very small mutagenic effect1. Conceivably, this reaction may be useful for the production of virus antigens. Biochim. Biophys. Acta, 123 (1966) 17-25

25

OXYGEN EFFECT OF HYDROXYLAMINE ON D ~ A ACKNOWLEDGEMENTS

We want to thank Dr. E. K. F. BAUTZ for valuable suggestions.

REFERENCES i 2 3 4 5 6 7 8

9 io ii 12 13 14 15 16 17 18 19

E. B. FREESE AND E. FREESE, Proc. Natl. Acad. Sci. U.S., 52 (I964) 1284. E. FREESE AND E. B. FREESE, Biochemistry, 4 (1965) a419. E. FREESE, E. BAUTZ AND E. B. FREESE, Proc. Natl. Acad. Sci. U.S., 47 (1961) 845. E. FREESE, E. B. FREESE AND E. BAUTZ, J. Mol. Biol., 3 (1961) 133. H. SCHUSTER, J. Mol. Biol., 3 (1961) 447. D. W. VERWOERD, H. I~OHLHAGE AND W. ZILLIG, Nature, 192 (1961) lO38. W. TROLL, S. BELMAN AND E. LEVINE, Cancer Res., 23 (1963) 841. A. BENDICH, E. BORENFREUND, G. C. KORNGOLD, M. KRIM AND M. E. BALIS, Acidi Nucleici e Loro Funzione Biologica, I s t i t u t o L o m b a r d o di Scienze e Lettere, Tipografia Succesori Fusi, Pavia, 1964, p. 214-237. W. ZILLIG, D. W. VERWOERD AND H. KOHLHAGE, Colloq. Intern. Centre Natl. Rech. Sci., Paris, lO6 (1962) 229. D. M. BROWN AND P. SCHELL, J. Mol. Biol., 3 (1961) 7o9. D . M . BROWN AND J. H. PHILLIPS, J. Mol. Biol., i i (1965) 663. H. SCHUSTER AND W. VIELMETTER, J. Chim. Phys., 58 (1961) lOO5. S. P. CHAMP AND S. BENZER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 532. E. FREESE AND H. B. STRACK, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1796. H. B. STRACK, E. B. FREESE AND E. FREESE, Mutation Res., I (1964) IO. J. H. PHILLIFS, D. M. BROWN, R. ADMAN AND L. GROSSMAN, J. Mol. Biol., 12 (1965) 816. R. G. WILSON AND M. J. CAICUTS, J. Biol. Chem., 24I (1966) 1725. N. I{. KOCHETKOV, E. I. BUDOWSKY AND R. P. SHIBAEVA, Biochim. Biophys. Acta, 87 (1964) 515 • H. SCHWEITZ AND D. LUZZATI, J. Chim. Phys., 6o (1963) I 173.

Biochim. Biophys. Acta, 123 (I966) 17-25