The Mutagenic Action of Hydroxylamine

The Mutagenic Action of Hydroxylamine J. H. PHILLIPS' AND

D. M. BROWN

University Chemical Laboratory, Cambridge, England I. Introduction . . . . . . . . . . . 11. Genetic Background . . . . . . . . . . 111. General Chemistry of Hydroxylamine Action . . . . A. Reaction with Nucleic Acids . . . . . . . B. Reaction with Cytosine Derivatives . . . . . C. Reaction with Uracil Derivatives . . . . . . IV. Experimental Investigation of Hydroxylamine Mutagrnesis A. The Model . . . . . . . . . . . B. Reaction of Hydroxylamine with Pols C . . . . C. Experiments with RNA Polymerase . . . . . D. Mechanism of Erroneous Replication . . . . . V. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

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349 350 353 353 354 356 358 358 359 361 362 364 366

1. Introduction I n the field of chemical mutagenesis, uncertainty holds sway. Despite the efforts of many investigators, success in clarifying the chemical events leading to mutation has, in general, been very limited. I n the absence of means for making direct observations on the genomic alteration that gives rise to the mutant progeny, a compromise position has to be taken, the degree of certainty regarding the mutagenic mechanism in question being judged by the convergence of a number of lines of evidence. The reasons for the difficulties inherent in the subject are not far to seek and have been discussed by others (I,,%?). From the chemical standpoint, the problems posed by reactions involving macromolecules are greater than might have been anticipated, and the difficulties in extending the details of apparently well-defined chemical reactions have proved formidable. One of the main stumbling blocks has been the 1 Preseiit address: Depart.mertt. o f Biochemistry, M:ikerere L1tiiversit.y College, Kampala, Uganda. 349

350

J. H. PHILLIPS AND D. M . BROWN

question of specificity. Specific reagents producing single base changes in DNA are difficult t o find and, indeed, only one possible candidate, methoxyamine, has been investigated a t all. Use of this reagent as a mutagen has been very limited although the chemically related hydroxylamine, which appears to be highly specific for the base transition C + T under the conditions normally employed for its use, is one of the more commonly used mutagens and has been investigated in detail. Indeed, its mutational specificity is such that it is the only mutagen to have been used confidently in codon assignment (see chapter by Woese in this volume) (3, 4, 4a). Hydroxylamine therefore, is a good choice for detailed chemical investigation. The objective of the work covered in this review has been to elucidate completely the mechanism by which hydroxylainine induces mutations upon interacting with the nucleic acid of a bacteriophage particle or with transforming DNA. In brief, our own and other work began with base analyses of treated DNA and examination of the reaction with the individual bases and nucleosides. It was then extended to polynucleotides and an in vitro polymerase system was used as a model for replication-this, in effect, providing a relay between the mutational studies themselves and the gross chemistry exhibited by the reagent. The latter phase is as yet incomplete, nor have the techniques developcd been extended to biological systems. Incomplete, too, are the physicochemical studies of the hydroxylamine reaction products, on which a detailed understanding of the error-induction in replication must depend.

II. Genetic Background Chemical mutagenesis of the rII region of bacteriophage T4 has been reviewed by Krieg ( 5 ) . However, a number of points of general importance with regard to hydroxylamine mutagenesis emerge also from studies of other bacteriophages and of transforming DNA. It causes mutations in bacteria (6),a yeast ( s a ) , and the fungus Neurospora crassa ( 7 ) . The mechanism whereby hydroxylamine induces abnormal chromosome patterns in mammalian (8, 9) and plant (10)cells is quite unknown and is not discussed in this review. Hydroxylainine has both strong inactivating and strong mutagenic effects on infectious nucleic acids. For the most part, these have been investigated separately, and the characteristics of the latter are known much more completely than those of the former. Freese and his coworkers have carried out systematic studies of the effects of hydroxylamine on T4 (11, fa) and Bacillus subtilis transforming DNA (1315) [being, in fact, the first to give an account of the mutagenic effect of the reagent ( f l ) ] , and other detailed analyses have been made by

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351

Schuster (16, 1 7 ) , Benzer (18), and Tessman (19), and their collaborators. Detailed analyses of relatively few hydroxylamine-induced T4 mutants have been published. However, the data available from both forward mutation (19) and hydroxylamine-induced reversion (11, 18) clearly indicate that the reagent induces a single class of base-pair transition ( 5 ) ; the same is found in the case of induction of mutations in transforming DNA ( 1 3 ) . Study of chemical mutagenesis was greatly refined by Tessman, however, by the use of the single-stranded coliphage S13 (19).The phage is exceedingly small (it is closely related t o bacteriophage pX174, which has only about 4500 nucleotides) , and consequently, unlike T4, it presumably has no dispensable genetic regions. Forward and reverse mutants were selected by host range. Systems were developed that gave unambiguous results and were probably free from the difficulty of distinguishing suppressor mutations, a difficulty that is inherent (although surmountable) in studies with T4. It could be clearly demonstrated from the mutation data that hydroxylamine induces a single class of base transition (out of the four possible classes) ; from chemical data (vide infra) this change is taken to be C + T (i.e., G - C+ A - T for a double-stranded genome). [We may note that, in contrast t o the clcarcut results with hydroxylamine, a number of other mutagens appeared to induce all the possible transition classes (19, 20).It seems very probable that to some extent this must be a reflection of secondary effects (for example, a replication error during repair of the lesion) , necessarily obscuring the site and nature of the primary event (Z).] There is good evidence from an analysis ( 6 ) of the reversion data of Champe and Benzer (18) that in T4 there is a close correlation between mutagenesis by hydroxylamine and by 5-bromodeoxyuridine, although we should note that an opposite conclusion might be drawn from the results of a clean-growth mutagenesis experiment performed by Terzaghi e t al. ( 2 0 ~ )However, . the situation seems to be quite the opposite for 513 mutants (21),even when the same host strain of Escherichia coli is used. This may reflect some special response of the phage-induced DNA polymerase to the presence of base analogs. Mutants induced by hydroxylamine in T4 appear to be widely distributed over both rIx cistrons (12, 18). Although no particular evidence for “hot spots” emerges from the mapping data [as found also by Tessman for S13 ( 1 9 ) ] the , rates of hydroxylamine-induced reversion a t different sites are in fact found to vary considerably (18). Drake, in his study of ultraviolet light-induced T4 mutants ( 2 2 ) ,found that many mutants reverting under the influence of base-analogs do so by suppressor mutations ; hydroxylamine, however, rarely induces such suppressors : this suggests the possibility that while very many G - C sites are suscep-

352

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H.

PHILLIPS AND

D. M. BROWN

tible to mutation by a base-analog such as 5-bromodeoxyuridine, very many sites may in fact be refractory to hydroxylamine mutagenesis. The degree of secondary structure of the genetic material appears to be of critical importance. Freese pointed out that T 4 is a thousand times more mutable by hydroxylamine than is transforming DNA (13, 2 3 ) ; denatured DNA is even more subject to mutagenesis. Native transforming DNA can be made more susceptible by changing the solvent, especially by adding ethylene glycol, which reduces the degree of secondary structure of the polymer. This difference of susceptibility has recently been utilized by E. S. Tessman to provide a precise method of estimating the amount of the double-stranded replicative form from S13 DNA in a mixture with single-stranded infectious DNA, based on the fact that hydroxylamine may be used to inactivate the latter essentially selectively ( 2 4 ) . It seems clear that effective hydroxylamine mutagenesis requires that the genome contain rather exposed bases. Studies of the p H dependence of the mutagenic change are of considerable importance in understanding its mechanism (see Section IV, D ) . Induction of mutants by hydroxylamine at p H values between 6 and 9 has been tested with T4 (16, 18) 513 (19),and tobacco mosaic virus RNA (17 ) . The very much reduced mutation rate a t higher pH values has provided the main evidence to support the idea that hydroxylamine interacts with cytosine rather than thymine in the genome. Especially interesting, however, are the few experiments examining the effect of p H values lower than 6. Two separate effects seem to be indicated. Schuster and Vielmetter, in experiments with T 4 in which the pH was reduced as low as 4.5, found a definite maximum a t p H 6 (16). On the other hand, with transforming DNA a t high temperature, Freese and Strack found that mutagenesis was much more effective a t p H 5.5 than a t 6.2 (13) and, although it is difficult to combine the results in their two papers, the later data of Freese and Freese (15)suggest that mutagenesis a t as low pH as 4.2 may be considerably more effective than a t pH 5.5. More complete data on these effects is very desirable, and it would be most interesting to have comparable data for S13. Schuster and Vielmetter made a further point with regard to T4 mutagenesis, namely, that treatment of the phage particles a t several p H values (no values were given) after removal of hydroxylamine had no effect on the mutation rate (16). They concluded that the primary action of the reagent with the hydroxymethylcytosine residues is responsible for the mutagenic change. The possible implications of these observations are discussed in Section V. Hydroxylamine is a convenient mutagen for study in the sense that, despite the complications in determining its mechanism, it seems to produce rather specific mutagenic changes, acting as a reagent for naked

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nucleic acid or phage particle in vitro. In vitro mutagens have considerable advantages over in vivo mutagens as is seen immediateIy when the effect of hydroxylamine on phage-bacterium complexes is investigated. The specificity of the reagent seems to be entirely lost (.25),presumably because of the many effects of hydroxylamine on the metabolism of the host cell and the possible further effects this may have on the bacteriophage replication. The possible complication in the interpretation of mutagenic data due to the presence of repair mechanisms in the host cell that reduce damage to the chromosome has already been mentioned, although more recent experiments (25a) suggest that this may not apply to hydroxylamine-induced mutagenic alterations in T4. The inactivating effects of hydroxylamine are not understood in any detail. The effect decreases a t relatively high hydroxylamine concentrations (above 1M ) (12, 15) , a phenomenon found both with bacteriophages and transforming DNA; there is little inactivation a t low pH ( 1 7 ) . N-Methylhydroxylamine, CH,NHOH, is completely similar to hydroxylamine in both mutagenic and inactivating effects on transforming DNA (15) although it does not seem to be mutagenic for T4 (1.2). Methoxyamine, NH,OCH,, on the other hand, is mutagenic in both systems (15, 26) but does not have an inactivating effect on transforming DNA. Light has been shed on the whole process of inactivation by the recent experiments of Freese and Freese (27), who observed that inactivation is decreased under anaerobic conditions and correspondingly increased in the presence of oxygen. Free radicals are generated under aerobic conditions, and doubtless these are involved in the inactivation process, since this, but not the mutagenic action, is diminished by radical inhibitors, such as pyrophosphatc.

111. General Chemistry of Hydroxylamine Action A. Reaction with Nucleic Acid,s

Hydroxylamine, although not very basic (pK, 5.96) (28), is a powerful nucleophilic agent, for reasons still not entirely clear. Although the purine bases are not readily attacked by nucleophiles, cytosine and uraciI derivatives are quite susceptible (29, SO). Extensive treatment of salmon sperm DNA with hydroxylamine followed by base analysis reveals a progressive loss of cytosine and a very much smaller loss of thymine; other bases are unaffected (31, 3 2 ) . I n place of cytosine, another base, later identified as N4-hydroxycytosine, is detected. Investigation of the action of hydroxylamine on individual bases, nucleosides, and nucleotides using chromatographic and spectrophotometric methods to follow the course of the reaction confirms that the pyrimidine, but not the purine, bases are attacked (11, 31-35).

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J. H. PHILLIPS AND D. M. BROWN

Furthermore, these studies reveal a marked p H dependence, cytosine derivatives reacting rapidly a t pH 6, uracil derivatives a t p H 10 ( 3 5 ) . Thymine derivatives react slowly but detectably under very forcing conditions (32), for example, heating in anhydrous hydroxylamine. By comparison with the free base itself, cytosine residues in native DNA react extremely slowly (32, 3 6 ) .

B. Reaction with Cytosine Derivatives We discuss here the chemistry of the reaction of cytosine and its derivatives (I) with hydroxylamine in strong aqueous solution. The p H optimum appears to be around 6.5, i.e., near the pK, of the reagent.

0A

N5N R I

NOH

IV

0

R

II

NHOH

I

III

With cytosine (I; R = H) itself, essentially complete disappearance of ultraviolet absorption in the 270 mp region occurs and a product having A,,, = 220-225 mp is formed with the structure 111. Although this compound may decompose in a number of ways a t pH values away from neutrality, it is stable for some hours in neutral aqueous solution a t room temperature (37). Strong acid converts it rapidly and quantitatively to N4-hydroxycytosine (IV), as does heating the dry cornpound. It is significant that IV is converted to 111 by aqueous hydroxylamine, but a t a rate that is a t most a fifth of its rate of formation from I. It follows that the major pathway from I to I11 does not include I V but instead must involve the intermediate I1 (46'). This intermediate cannot be detected in the reaction with cytosine or cytidine 2'(3')-phosphate so that clearly I + I I is the rate-limiting step. However, when polycytidylic acid is used, the intermediate I1 is stabilized to a small extent and its presence in the polymer can be demonstrated ( 3 8 ) .

355

MUTAGENIC ACTION O F HYDROXfLAMINE

The mechanism of the niajor reaction of hydroxylamine with cytosine, and, indeed, with all of the A']-substituted derivatives studied, (e.g., I in which R = Me, ribosyl, 2'-deoxyribosyl, ribosyl 2'(3') -phosphate) (3.2, 38) is thus a conjugate addition of hydroxylamine leading to saturation of the 5:6 double bond followed by a rapid exchange of the amino group by hydroxylamine. The second step is an example of a very general displacement reaction: 5,6-dihydrocytosines (V) are very readily hydrolyzed to dihydrouracils (39) and they react extremely rapidly with hydrosylamine to yield i~'-liytlrosy-5,6-dihydl.oeytosincs (VI) (40) and with other nitrogen nuclcophiles to yield analogous products (40, 41 ) .

V

VI

At this point we should note briefly that methoxyamine reacts with cytosinc derivatives analogously to hydroxylamine although with a lower pH optimum ( 3 2 ) .No reaction has been demonstrated with uracil derivatives (4%'). The adduct formed between N-methylhydroxylamine and cytosine, viz. that corresponding to 111, is very unstable, and the exchange product, N'-methyl-N4-hydroxycytosine, is isolated from the reaction mixture (@). There are two positions on the cytosine ring a t which nucleophilic attack can occur. The C-6 position has already been discussed. Displacement reactions a t (3-4 are also known, exemplified by the very slow base-catalyzed hydrolysis to uracil derivatives (44). [The deamination observed in mild acid solution may depend on reversible addition of a nucleophiIe to the 5,6-double bond and hydrolytic displacement of the amino group in the internietliate (45j.l Janion and Shugar (46a) were the first to point out that the reaction between hydroxylamine and 5-hydroxymethylcytosine (VII) leads to the exchange product VIII. NOH

VII

VIII

They found no evidence for the presence of an intermediate corresponding to 111 and suggested that a direct displacement occurred. Present

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J. H. PHILLIPS AND D. M. BROWN

kinetic evidence (do), however, does not distinguish between such a displacement (VII + VIII) and the formation of a 5,6-adduct that undergoes the displacement reaction followed by a rapid elimination from the 5,6-positions. The same is true for !j-methylcytosine, and in each case the reaction proceeds more slowly than with cytosine. More recently, Lawley TABLE I RATECONSTANTS FOR REACTIONS BETWEEN HYDROXYLAMINE A N D CYTOSINE DERIVATIVES Compound Cytosine (I; R

=

H)

2'-Deoxycytidine (I; R

=

2'-deoxyribosyl)

5-Hydroxymethylcytosine (VII) a In 3.5 M hydroxylamine, pH 6.5, 35°C. Data from Brown and Hewlins (40).

b

k

Reaction

(hr-I)

I-+ I1 I+IV IV -+ I11 I11 --+ Iv I --+ I1 I + N VII + VIII

4.5" 0.9"

0.2

0.02b

1.25O 0 . 25a 0.3"

In 4.0 M hydroxylamine, pH 6.5, 35°C.

(47') has obtained kinetic evidence that some N'-hydroxycytosine (IV) is formed simultaneously with I11 in the reaction of hydroxylamine with cytosine, and this has been confirmed in other work (40) that shows, in addition, that the back reaction (111 + IV) also contributes to its formation. Constants are given in Table I by which the rates of the various reactions can be compared. C. Reaction with Uracil Derivatives As mentioned above, hydroxylamine reacts with uracil derivatives, albeit to a much smaller extent than with cytosine derivatives, and the gross chemistry of this reaction has been elucidated by two groups (33, 3 5 ) . Examination of reaction profiles as followed by absorbance decrease (Fig. 1) shows that the initial rate of reaction with uracil derivatives is in fact fairly high, even a t pH values below neutrality, although the reaction does not proceed so far toward completion a s in the cytosine case. Thymine, by this criterion, appears to be essentially unreactive (11, 33). However, we detail here briefly the course of reaction with uracil, in view of its relevance t o the question of hydroxylamine reactivity and to its possible usefulness for modifying nucleic acids before enzymatic cleavage, (34) or as a probe for studying the function of specific bases, as applied recently to transfer RNA [e.g., (48-60)] and to synthetic messenger RNA's (61).

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MUTAGENIC ACTION OF HYDROXYLAMINE

I

_--I

..

FIG. 1. Reaction with 2.5 M hydroxylamine hydrochloride, p H 6.7, 22"C, followed by decrease of optical density a t A.,

The shape of the reaction profile is indicative of a relatively rapidly attained equilibrium (IX X) . The reaction probably then proceeds by a mechanism analogous to hydrazinolysis (52). Following addition to the a,P-unsaturated carbonyl system (IX + X) ring closure occurs with formation of the 3,4-dihydro-3-ureidoisoxazo1-5-one(XI). Analogous pyrazolone derivatives have been isolated as hydrazinolysis intermediates ( 5 3 ) . The isoxazolone (XII) is liberated by elimination of urea. It may be assumed that adducts of type X are rather labile with respect to elimination of hydroxylamine ; under basic conditions the ring

Ix

X

1

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J. H. PHILLIPS AND D. M. BROWN

closure is facilitated. This explains the base-catalysis observed. The rate decreases above p H 10, which is above the pK, of the uracil derivative, so that the initial nucleophilic addition is hindered. It thus seems that the equilibrium (IX e X) is rapidly established and is slowly displaced by irreversible isoxazolone formation as evidenced by the slow decrease in optical density that is observed. The adduct is unstable with respect both to reversal to the parent uracil and to isoxaxolone formation and presumably has a relatively short-lived existence. 5-Substituted uracil derivatives are generally unreactive, and this applies even to the electrophilic 5-nitrouracil, although this may reflect, in the latter case, simpIy an unfavorable equilibrium position in the addition reaction. The rapid reaction observed between hydroxylamine and 5-bromouridine (Fig. 1) (11, 4.3) is therefore unexpected, the more so as 5-bromocytidine reacts very slowly (4.3). I n the reaction with 5-bromo-l-methyluracil, bromide ion is liberated, the non-ultraviolet-absorbing component of the equilibrium mixture being, in fact, the adduct (X; R = Me) and 3-methyluracil is one of the products of the reaction. The reaction has a sharp pH-rate profile with a maximum a t pH 7.2 ( 4 3 ) . The high rate of the reaction, even faster than that of cytosine a t comparable pH values, suggests that the decomposition of the bromouracil residues causes the rapid inactivation of bacteriophages observed when thymine is replaced by 5-bromouracil (If).

IV. Experimental Investigation of Hydroxylamine Mutagenesis

A. The Model Modern biology is clearly a science of models. Whether conceived or built, they are connected to actual biological proccsses by reason and rarely by experirncnt ( 5 4 ) . Bacteriophage mutagcnesis, itself of interest as a model for studying genetic processes in higher organisms, must be imitated by a simpler model if a full understanding of the process on the molecular level is to be gained. The primc requirement of the model is that every mutational event be scored. Mutagenesis of even the well-defined rII region is impossibly ill-defined: not only is the genome a complex DNA helix but the phenotypic expression of mutations is inevitably largely prevented ( 2 ) . For obvious reasons, we, in our own work, decided to study the action of hydroxylamine on the readily available homopolymers, especially poly C. It is not immediately obvious that this polynucleotide, forming :L single-stranded structure with stacked bases a t p H values near neutrality (55, 5 6 ) , is a good model for the DNA molecule, the main contrast

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being the nature of its secondary structure which seems to result mainly from the hydrophobic interactions of the bases (56, 57). A t first sight, a double-stranded structure such as poly I.poly C would appear to have greater relevance, for it is by no means certain that patterns of reactivity are the same when a particular base is situated in such differing environments. This problem has been studied principally with reference to photochemistry. For example, cytosine hydrate formation occurs on irradiation of poly C but not of the complex poly 1-poly C (58) or of poly dI-poly dC ( 5 9 ) , and hydrate formation is much reduced in poly Aepoly U compared with poly U (60). I n this connection, we may refer back t o the work of Freese et al. (12, 1.3, 15) on hydroxylamine-induced mutagenesis in bacteriophages and B. subtilis transforming DNA. From a consideration of mutation rates in various solvents and especially in aqueous glycol solutions it was concluded that the mutagenic reaction was strongly dependent on the exact structure of the DNA (IS). The contribution of the hydrophobic forces to the secondary structure of DNA is a matter of present dispute (61), but ethylene glycol presumably produces an unwinding effect. Bacteriophages were found to be one thousand times more mutable than transforming DNA under similar conditions (1.0M hydroxylamine in 1.3 M NaCl a t pH 7.5 and 37") ( I S ) ; the rate of induction of mutations in phage is intermediate between those of native and denatured transforming DNA, but the activation energy is similar to that for denatured DNA (14). A possible conclusion from these experiments was that the phage DNA was distorted by packing, rendering some regions more accessible to the mutagen (cf. also 61a,b). [One may recall Drake's suggestion from genetic evidence that not all C - G sites are equally accessible to hydroxylamine (Section 11).] It would thus appear that the comparatively exposed poly C molecule may in fact be a better model for in vitro mutagenesis than a highly structured complex such as poly I.poly C. Poly 5-methylcytidylic acid has recently become available (62). It has a greater degree of secondary structure than poly C and is clearly a better model for bacteriophages containing 5-hydroxymethylcytosine, such as T4 (46).

B. Reaction of Hydroxylamine with Poly C Reaction of poly C with hydroxylamine is considerably slower than that of cytidylic acid. I n excess reagent, the reaction proceeds with first-order kinetics, and analysis of the partly reacted polymer shows that the appearance of the N4-exchange product (111) is considerably delayed (38).Depending on whether this is consequent on the polyanionic nature of the substrate or steric hindrance, which affects C-4 and

360

J. H. PHILLIPS AND D. M. BROWN

C-6 differentially, denatured DNA may or may not show the same effect. There is a certain limit to the amount of useful information that may be derived from large extrapolations from relatively insensitive absorbance measurements. Using methoxyamine-C1* it is possible to measure the product content of the polymer a t very small extents of reaction ( 6 3 ) . Figure 2' shows the formation of products I1 and 111 in

2.o

D

I .5

e

c

0

u)

3

U .-

; 1.0

c

W

cn

0 * c 0

E

n

0.5

Hours of reaction with NH,OCH,

FIG.2. Reaction between poly C and 1.0M methoxyamine hydrochloride, pH 5.5, 37°C. Curve A : percentage of cytosinr residues rcarted, curve B: percentage residues (111, residues converted to N*-methoxy-5,6-dihydro-6-m~thoxyaniinocytosine sre text) ; curve C : percentagc. of residues converted to 5,6-dihydro-6-methoxyaminorytosine residues (11). [From the data of Phillips et al. (G3.1

the polymer for up to 2% of reaction. The intermediate adduct (11) reaches a low steady state concentration, although how long this is maintained cannot be deduced from the data. The product (111) may contain a small proportion of IV, possibly up to one-fifth ( 4 7 ) , since this is not distinguished by the assay. Concomitant with the reaction, there is a loss of secondary structure of the poly C. This presumably results directly from the saturation of

MUTAGENIC ACTION OF HYDROXYLZAMINE

361

the 5,6-double bond and the consequent lack of interaction of the affected base with its neighbors. An analogous situation is found when uracil residues in poly U undergo reduction (64).

C. Experiments with RNA Polymerase Grossman e t al. first used highly purified RNA polymerase from Micrococcus Iysodeikticus to investigate mutagenic effects by examining the effect of ultraviolet irradiation on the template properties of poly C (65). The same system has been used to investigate the effects of hydroxglamine. The enzyme used is thought to be that involved in the synthesis of RNA from a DNA template in vivo (66) ; as it can also utilize homoribopolymers as templates for the synthesis of complementary polynucleotides ( 6 7 ) ,it is a convenient model for the replication process. The clear advantage of this system as a model is that the “mutagenic” events in the template poly C should be detected by and relatable to adenylate incorporation in the poly G synthesized (if the theory of transitions is correct) without the difficulties due to code degeneracy in scoring methods relying on translation (such as identifying mutant proteins or use of an in vitro protein synthesizing system). The RNA polymerase appears t o “read” the template accurately and sequentially (68). When the template is poly C, one equivalent of poly G is formed in the presence of M . Eysodeikticus RNA polymerase and G T P (69).This is firmly complexed with the poly C, and the product does not act as a template for further guanylate incorporation. Treatment of poly C with hydroxylamine reduces its capacity to act as a template for poly G synthesis. This capacity is partially restored if ATP, but not CTP or UTP, is added to the reaction mixture: adenylate is incorporated into the newly synthesized poly G, primarily as single residues flanked on both sides by guanylate residues, as shown by nearest-neighbor analysis (70, 7 1 ) . Treatment of poly A, however, has relatively little effect on its ability to act a s a template for poly U synthesis. Treatment of poly IJ at pH 6.5 leads to some decrease of its template activity; however, this cannot he recovered by addition of a second nucleoside triphosphate. These experiments fend strong support to the original contention that hydroxylamine may act as a mutagen by altering a cytosine residue so that it is replicated as if it were thymine. The next problem is the determination of which possible product of hydroxylamine action directs adenylate incorporation and how this erroneous incorporation occurs. The kinetics both of guanylate incorporation in the presence of ATP and of the adenylate incorporation itself as a function of time of hydroxylamine treatment of the poly C strongly suggests that residues of 11,

362

J. H. PHILLIPS AND D. M. BROWN

the mono-adduct, rather than 111 in the polymer are responsible for the “mutagenic” error (70). As noted before, methoxyamine is a mutagen and in its reactions with cytosine derivatives is similar to hydroxylamine. Use of the CI4-labeled compound has two advantages; first it is possible to determine the proportions of each reaction product in the poly C after very small extents of reaction (Fig. 2) and, secondly, the number of alterations in the template necessary for each adenylate incorporated can be derived. These experiments (63) strengthen the case that, in this model system, 5,6-dihydro-6-hydroxylaminocytosineresidues (11) are replicated like uracil residues and indicate that each residue of this base in the template is replicated in this way.

D. Mechanism of Erroneous Replication The various theories that have been entertained for the explanation of hydroxylamine mutagenesis are indicated in Fig. 3. At present, mechanisms A and C are favored and we present here the evidence in their favor and against the others. Deamination to uracil or its adduct (D and E) seemed attractive hypotheses a t first (33) in view of the known lability of dihydrocytosines toward hydrolysis. However, analyses of even extensively hydroxylamine-treated poly C have failed to reveal any uridylate residues in the polymer (38),and the recent studies of Johns et al. on the deamination of 5,6-dihydro-6-hydroxycytosine (7%’) render it most unlikely that deamination could occur under mutagenesis conditions. I n the case of the poly C-RNA polymerase model system, both C and F are excluded because of the scarcity of these species in the poly C template while adenylate residues are being incorporated (63). They could be involved only if each altered base directed the incorporation of a t least two or three adenylate residues. The experimcnts with methoxyamine quoted above also excluded B as a possibility. From the estimated pK, of 11, taken to be close to 5.6, as found for the structurally similar cytosine hydrate (72), it is clear that not more than one in ten of the residues of I1 in the poly C could be protonated. Yet every residue is, in fact, replicated by adenylate incorporation. One is therefore brought to a consideration of mechanism A. The induction of errors in the replication process leading t o transition mutations has, until now, been discussed solely in terms of changes in the ionization level or tautomeric state of the altered base (73) although, doubtless, a more sophisticated view will be developed ( 7 4 ) . We have rejected mechanism B, based on a change in ionic state. The tautomeric constant of I1 is unknown. The related 1-methyl-5,6-

363

MUTAGENIC ACTION OF HYDROXYLAMINE

H

I

HO.NH

m FIG.3. Possible mechanisms for base-pairing with adenine in hydroxylamine mutagenesis.

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dihydrocytosine (V: R = Me) has a tautomeric constant of ca. 25 in favor of the amino form in aqueous solution (40)~compared with cytosine in which KT N lo5 (75). The less polar imino form predominates in a medium of low dielectric constant, a situation that may correspond to the conditions a t the point of replication. Indeed, a tautomeric constant of 0.2 or less (i.e. KT N 5 in favor of the imino form), if it applied to 11, would adequately account for the incorporation results and support mechanism A. However, it must be emphasized that extrapolation from such results to the case in point is a matter of considerable uncertainty. We are compelled to reconsider the question of how far the poly C model represents the case of mutagenesis itself. A good case may be constructed for supposing that for bacteriophage T4, a t least, mutagenesis is mediated by mechanism C. As noted before, reaction of aqueous hydroxylamine with 5-h.ydroxymethy1cytosine leads to N4-hydroxy-5-hydroxymethylcytosine as the only detectable product (46), although we have no information on the degree to which the addition and exchange reactions are affected by secondary structure in DNA, relative to one another. Moreover Janion and Shugar (46) have pointed out that if hydroxylamine mutagenesis of transforming DNA is as strongly dependent on low pH as suggested by Freese e t al. (15),an explanation may be found in the acid-catalyzed elimination from residues of type I11 to yield a n N4-hydroxycytosine (IV) [cf. ($?)I. It is in any case clear that the poly C model is inappropriate as a test for mechanism C. However, copolymers of cytidylic and N4-hydroxycytidylic acids are available and act as effective templates for poly (A,G) synthesis in the presence of RNA polymerase (76). Furthermore the kinetic evidence indicates that N4-hydroxycytosine residues direct the incorporation of adenylate residues with high efficiency (>50%). Of some significance, too, is the finding that N1-substituted derivatives of N4-hydroxy- and N4-methoxycytosine are essentially completely in the oximino tautomeric form (IV) with KT of about 10 ( 7 7 ) .I n addition they show strong interaction with a 9-alkyladenine, but not with a 9-alkylguanine (40), in the system used by Rich e t al. (78). The specificity found in the hydrogen bond interactions of the normal base-pairs ( i e , G with C, A with U) (78-81) and the demonstration of bonding between N4-hydroxycytosine and adenine derivatives is strong presumptive evidence for error induction by mechanism C.

V. Conclusions Although a conclusive and unified theory for hydroxylamine mutagenesis cannot yet be presented, the two alternative possible mech-

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anisms (A and C) seem to be well defined and to lead to a number of clear predictions. We favor addition of hydroxylamine to cytosine residues to yield 5,6-dihydro-6-hydroxylaminocytosineresidues as the active error-promoting species in the case of single-stranded cytosine-containing bacteriophages such as S13, and possibly transforming DNA. At the moment the mechanism for T-even bacteriophages remains open although the chemical and enzymatic evidence strongly favors that in which N4hydroxy-5-hydroxymethylcytosine residues lead to replication error. It may be very pertinent in this connection that N-niethylhydroxylamine is mutagenic for transforming DNA but not for T4 (Section 11); the exchange product (XIII) cannot take up the oximino form nor is it likely to base-pair effectively, while the 5,g-adduct (XIV) should be insig-

XIII

XIV

nificantly different from the hydroxylamine adduct. It is, in any case, clearly possible that no single unique mechanism covers all cases of hydroxylamine mutagenesis. Further experimental work is clearly needed ; for instance, chemical and enzymatic investigations using other model systems such as poly5MeC and poly I =poly C or appropriate deoxyribonucleotide polymers. Chemical investigation of bacteriophages or DNA treated with labeled methoxyamine, further biological experiments on the effect of acid treatment on mutagen-treated material and mutagenesis under conditions producing defined chemical products arc still required. In conclusion, we draw attention to the possible beginnings of a rationale for cytosine-based mutagenesis. As we have indicated, there is strong evidence that 5,6-dihydro-6-hydroxylaminocytosine residues (XV) may be responsible for C + T transitions in some systems; furthermore, it has been suggested (82) that hydrazine-induced mutations, thought to be primarily transitions in the case of T4 (83),result from the reducing action of di-imide to yield the 5,6-dihydrocytosine derivative (XVI ; R’ = CH,OH) . Irradiation of bacteriophages yields a high proportion of transition mutants; in the case of T4 these are mainly G - C + A - T transitions ( 2 2 ) ,and the majority of mutants induced in 513 have definitely been identified as C + T (20). Taken in conjunction with experiments utilizing the RNA polymerase model system described above (66) and

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with Johns's experiments showing cytosine hydrate to be a primary product of thc irradiation ( 8 4 ) ,there seems good evidence that 5,6-dihydro-6hydroxycytosine residues (XVII) are responsible for the transition. Com-

xv

XVI

XVIII

XVII

plementary experiments have now been performed with R.NA polymerase in which it has been shown that 5,B-dihydroCTP (XVIII; R = ribosyl Y-triphosphate) may act as a substrate for the DNA-dependent enzyme, being recognized as either CTP or UTP ( 8 5 ) . It therefore seems reasonable to expect that some unity in this area of chemical mutagenesis will be established within the fairly near future. REFERENCES 1. L.

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