DNA base modification: Ionized base pairs and mutagenesis

Mutation Research, 177 (1987) 201-218 201 Elsevier

MTR 04329

DNA base modification: Ionized base pairs and mutagenesis

L a w r e n c e C. S o w e r s *, B a r b a r a R a m s a y Shaw, M a r t i n a L. Veig l a n d W . D a v i d S e d w i c k

Department of Medicine of Duke University Medical School and Department of Chemistry of Duke University, Durham, NC (U.S.A.)

(Received 12 April 1986) (Revision received 26 October 1986)

(Accepted 3 November 1986)

Keywords: DNA base modification; Ionized base pairs; Hydrogen bonding

Summary

The nature of hydrogen bonding between normal and modified bases has been re-examined. It is proposed that hydrogen-bonding schemes may involve tautomeric, ionized or conformational forms (syn, anti and wobble). Several important cases are presented or reviewed in which physical evidence indicates the existence of ionized base pairs. When thermodynamic values determined in aqueous solution under physiological conditions are considered, it can be argued that base ionization will contribute substantially to the stability of many biologically relevant base pairs containing modified bases. A significant incidence of ionized bases in DNA may have important kinetic ramifications for the further chemical reactivity of both the modified base and its cross-strand pairing partner. Moreover, DNA structure at and surrounding ionized base pairs may be altered. For this reason, the model presented in this study should be useful as DNA-sequence analysis becomes more commonly applied to the study of mutagenesis.

I. Introduction

Modification of DNA is the chemical basis of mutagenesis and also underlies the mechanism of many agents used in cancer chemotherapy. Many mutations arising from either chemical modification of DNA or incorporation of promutagenic base analogs are believed to result predominantly from aberrant hydrogen-bond formation. Several schemes have been proposed to account for the generation of base mispairs including tautomerization (Watson and Crick, 1953b; Freese, 1959;

* Present address: Department of Molecular Biology, Univer- sity of Southern California, Los Angeles, CA (U.S.A.)

Correspondence: W. David Sedwick, Ph.D,, Division of Hematology and Oncology, Case Western Reserve University Medical School, Cleveland, OH 44106 (U.S.A.).

Katritzky and Waring, 1962; Topal and Fresco, 1976a, b; Fresco et al., 1980; Singer and Kusmierek, 1982), ionization (Lawley and Brookes, 1961, 1962; Lowdin, 1965; Sowers, 1983; Sowers et al., 1984; Richards et al., 1984) and conformational changes such as ant i-syn purine rotation (Topal and Fresco, 1976a, b), and wobble base-pair formation (Crick, 1966).

This paper reexamines the nature of hydrogen bonding between normal and modified bases in DNA. Although all of the hydrogen-bonding schemes are expected to be in equilibrium with each other, a thermodynamic preference for formation of ionized base-pairs presents kinetic as well as structural possibilities that could have an effect on the chemical reactivity of both a modified base and its cross-strand pairing partner, the stability of DNA structures, and the processes of

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

202

repair for modified bases. Equilibrium constants for base ionization and tautomerization are em- ployed to argue the general importance of ionized structures in modified DNA and to illustrate a number of cases where such structures may be important under physiological conditions. We conclude that the mispairing potential of a wide variety of important mutagenic modified bases are consistent with formation of ionized base pairs. This concept constitutes a general model which facilitates explanation of the spectrum of base- pairing configurations induced both by incorporation of modified bases and by in situ base modification.

II. Development of the model

In 1953, Watson and Crick (1953a, b) presented their general model for the structure of the D N A molecule. This model suggested that the specific

formation of hydrogen bonds between purines and pyrimidines, within the steric constraints of the D N A helix, was the molecular basis of informa- tion transfer in biological systems. As a result, base-substitution mutation was ascribed to altered specificity of hydrogen-bond formation.

Altered specificity of hydrogen-bond formation may result from proton exchange. Proton exchange between the solvent water and hydrogen- bonding sites will yield ionized bases and proton exchange with electron lone pairs of the same molecule will give rare enol and imino tautomers. The structures of the normal DNA bases in the preferred, ionized and rare tautomeric conformations are shown in Fig. 1. The structures are presented in terms of the "pro ton code" devel- oped by Lowdin (1965). Fig. 1 illustrates that the tautomers and normal bases have the same number of protons for H-bond formation, but that these protons can be arranged so that they confer

I O N IC

H:L H

H R ~ :H

N O R M A L " H:

H :1 1~. H

cytos ine guanine

H

, : thymine a d e n i n e

T A U T O M E R :H

H: H

R 01:H

Fig. 1. The pairing configurations of the protons of the DNA bases in normal, ionic and rare tautomeric forms (cytosine, R = H; thymine, R = CH3).

a different "proton code" for base pairing. Alter- natively, nucleic acid bases can gain or lose a proton to form an ionized structure, providing yet another possible base-pairing configuration.

Ionization, tautomerization and conformational changes are intrinsic properties of nucleic acids. Fidelity of DNA replication, however, requires that one structure of each base be highly preferred under physiological conditions. Replication of DNA in the presence of bases with altered hydrogen-bonding characteristics will result in incorrect base insertion and genetic mutation. In the model presented here, potentially mutagenic base modifications are discussed in terms of their influence on the conformational equilibria of their base-pairing potential in DNA.

The tendency of a nucleic acid derivative to ionize at physiological pH can be quantitatively described by its ionization constant, or pK value. Several methods can be used to determine pK values of nucleic acid bases, most notably spectra- photometric (Shugar and Fox, 1952; Fox and Shugar, 1952; Lewin and Humphreys, 1966; Sowers, 1983) or potentiometric titration (Lewin and Humphreys, 1966; Christensen et al., 1967; Aylward, 1967). Based upon pK values, it can be determined that approximately 1/500 of each of the common bases will be ionized at physiological pH.

The tendency of a nucleic acid derivative to form its unpreferred tautomeric conformation, described quantitatively by its tautomeric equilibrium c o n s t a n t (Kt) cannot be directly determined. These calculations require locking a derivative into its rare tautomeric conformation by methylation as described previously (Angyal and Angyal, 1952; Kulikowski and Shugar, 1978). Be- cause the ionic configurations of both rare and preferred tautomers are identical, the ratio of molecules in the normal and rare tautomeric forms ( K t ) is equal tO the ratio of their ionization equilibrium constants as indicated in Fig. 2. The pK values of the imino tautomers of both cytosine (Brookes and Lawley, 1962) and adenine (Macon and Wolfenden, 1968) are above 8.5. Thus these tautomeric equilibrium constants for the normal bases are approximately 10 -5 (Kenner et al., 1955; Katritzky and Waring, 1962; Wolfenden, 1969; Topal and Fresco, 1976a, b; Fresco et al., 1980;

203

H \N-"

e(~3 N- H ^^ 'kN, 2.~/ _HZ+,J K CAL/MC)L R/ "°

TAUTOMERIC (imino) '+H

6.4 K CAL/MC)/ ;N_~ O

IONIZED /~~ - -H 41KCAL/MOL ( protonated amino )

R 0 PREFERRED(amino)

Fig. 2. Determination of the tautomeric equifibrium constants by the method of basicity. The free-energy difference between the amino, imino and protonated conformations of deoxycytidine determined from the p K of the normal deoxynucleoside (pK 4.3) and the p K of N3-methyl-2'-deoxycytidine (pK 8.8) which is equivalent to the imino conformation. Constants for the following equifibria were calculated: Total energy difference for protonation of the amino versus imino conformations from the relationship:

AG O= - R T In K T = +6 .4 kcal /mole

where

K T = [ imino] / [amino] = Kimin°/K amin°

= K - pK(imin°)/K- pK(amino) = 10- 8"8/10- 4.3 = 3.2 X 10 - 5

The free-energy difference between the neutral (amino and imino) and ionized forms at physiological pH is given by:

ZIG' = - R T In K ' =1.364 ( p K - p H ) at 25°C

where K ' = [neutral]/[ionized] at physiological pH, therefore

AG'~mi.o = 1.364 (4 .3-7.2) = - 4.1 kcal /mole

and

AG(mi.o = 1.364 (8.8 -- 7.2) = + 2.3 keal /mole

Singer and Kusmierek, 1982), which means that roughly one in one hundred thousand will be found in the rare tautomeric conformation under physiological conditions.

Another way of expressing equilibrium constants is in terms of free-energy differences, AG"

204

(Fig. 2). In this paper, a standard physiological state is used in which the standard state H + concentration is 6.3 × 10 -8 (pH 7.2) rather than one molar. The free-energy changes associated with base ionization at physiological pH for the normal bases are between 3 and 4 kcal/mole whereas free-energy changes associated with tautomerism is approximately 6 kcal/mole (Fresco et al., 1980). It is immediately apparent from this analysis that both ionization and tautomerization are disfavored events for monomers at physiological pH. It is also clear based on equilibrium arguments, however, that ionized forms of the normal bases are two orders of magnitude more common than are rare tautomers, with an energetic preference for ionization versus tautomerization at physiological pH of 2-3 kcal/mole.

The pK values of the DNA bases (Dunn and Hall, 1975) which act as hydrogen bond donors (guanine and thymine) are more than two pH

units above physiological pH whereas the pK values of the corresponding hydrogen-bond acceptors (cytosine and adenine) are two pH units below physiological pH (Fig. 3). Modification of DNA bases which causes pK values to approach physiological pH will increase the proportion of ionized bases. Increased ionization will cause potential hydrogen-bond donors to become acceptors and acceptors to become d o n o r s - resulting in an increase in potential illegitimate base-pair formation. Alternatively, base modification which pushes pK values in the opposite direction (away from physiological pH, i.e. toward more acid pKs for cytosine and adenine or toward more basic pKs for thymidine and guanine) will also have deleterious effects. Since the strength of the hydrogen bond is roughly proportional to the acidity of the hydrogen-bond donor and the basicity of the hydrogen-bond acceptor (Vinogradov and Linell, 1971), decreased ionization tendency at physio-

"0 o E

0 ~

~ L e- 0

"10

L

>

L

~ s i pK, 12 decrea n 9

hydrogen bond st rength

~ ,- 10 N - N NOR

i n c r ' e a s i n g - 9 ~//'/~ y d r o g e n b o n d s t r e n g t h :,:,:,:

increasing 8 ?///// ornbi uH.y

::::i::i:: increasing 7 :i:i:!: ambigu i ty ii!iiii . :!:i:i:i . " 6 :.:.:.: Increosln 9 ii!!i!!hydrogen bond., s%reng%h " " ' " ' :::::::: 5

:,:.2.2, ~ / N • ACCEPTOR • 4

decreasing hydrogen bond strength

1

physiological pH

GUANINE THYMINE

5 - bromouraci l

5 - f luorourac i l

guaninelcis-Pt

5 - met hy lcy tos ine 2 - aminopur ine CYTOSINE ADENINE

5- hydroxyrnet hylcytosine

5 - iodocytos ine

5 - b romocy tos ine

5 - f l uo rocy tos ine 06 aklylguanine

0 4 a lky l thymine

Fig. 3. This figure illustrates the potential consequences of base modification in terms of the base-pairing potential of a number of modified bases. As indicated, hydrogen-bonding potential of donor and acceptor sites can be reversed or rendered ambiguous by base modification. In the examples, the hydrogen-bonding potential of each DNA base as donor or acceptor at physiological pH is depicted as a function of the p K value of its central hydrogen-bonding site.

logical pH will result in weaker hydrogen bonds. The stability of the double-stranded DNA molecule is in part dependent upon hydrogen bonding, but further, the fidelity of DNA replication is largely dependent upon differences in hydrogen- bonding free energy between "right" and "wrong" base pairing possibilities (Galas and Branscomb, 1978; Clayton et al., 1979; Goodman et al., 1980). Reduction in the strength of hydrogen bonds (due to altered pK values of modified bases) may reduce discrimination during DNA replication. It is tempting to speculate that the pK values of the naturally occurring DNA-base hydrogen-bond donor and acceptor sites, symmetrically disposed with respect to physiological pH, represent an evolutionary optimum for counterbalancing the requirement for hydrogen-bonding strength and the problem of base-pairing ambiguity which may result from ionization. In the following discussion, we will present evidence that alteration of pK values of potential hydrogen-bonding sites may explain the mutagenicity of many modified bases (Fig. 3).

When moving from solution into the DNA helix, one must account for stability factors which comprise DNA structure. First, sites of ionization of the DNA bases (proton donor and acceptor sites) are involved in hydrogen-bond formation in Watson-Crick base pairing. In normal base pairs, protonation of either cytosine or adenine would disrupt hydrogen bonding, and ionization of either thymine or guanine would result in the loss of one hydrogen bond. Because ionization of normal Watson-Crick bases would result in loss of hydrogen bonds, base-pair formation generally acts to suppress ionization (von Hippie and Printz, 1965; Jordan, 1955).

In several cases important to mutagenesis, however, base ionization may restilt in the formation of additional hydrogen bonds. Therefore, hydrogen-bond formation may provide the driving force for base ionization at physiological pH. Below, several cases will be presented in which ionized base pairs have either been demonstrated, are implied in solution or would be expected.

III. Ionized base pairs in DNA

The examples presented below are illustrative of the argument that ionized bases are a common

205

consequence of mutagenic base modification. In some cases, these interactions may stabilize a mutagenic base-pairing combination or as in many of the examples below, ionized bases may promote the most stable configuration of a modified base with its unmodified pairing partner prior to ini- tiation of a DNA-repair process.

(,4) Hydrogen-bond formation may drive protonation

Bicytosine cation. The pK values for naturally occurring cytosine derivatives in solution are generally between 4 and 5, indicating that protonation would be thermodynamically disfavored at physiological pH. However, if cytosine residues form an additional hydrogen bond as a consequence of protonation in a polymer structure, the unfavorable free-energy change associated with protonation could be largely offset. Hydrogen- bonding of a protonated cytosine residue has been observed in the formation of the "acid structure" of polyribo- and deoxyribo-cytidylic acid as shown in Fig. 4. This structure, involving a shared proton (proton bond) between two cytosine residues (bicytosine cation), has been supported by crystallographic * (Marsh et al., 1962; Langridge and Rich, 1963; Westhof and Sundaraligam, 1980; Cruse et al., 1983), spectrophotometric (Akinrimisi et al., 1962; Inman, 1964; Zmudzka et al., 1969; Gray et al., 1980) and other physical data (Montenay- Garestier and Helene, 1970; Langer and Golan- kiewicz, 1978; see refs. in Saenger, 1984 for discussion). An alternative protonated C structure has been proposed by Gray et al. (1980).

The complex pK value associated with the helix-coil transition of a dC polymer is 7.4, well above the pK of the monomer (Inman, 1964). At pH 7.4, half of the cytosine residues are proto-

It should be pointed that the protonated fold-back helical structure of crystalline polymers of polycytosine (Marsh et al., 1962; Langridge and Rich, 1963) has been disputed (Arnott et al., 1976; Leslie and Arnott, 1978; Saenger, 1984, for a review). However, the hemi-protonated cytosine dimer has been shown to exist in crystals (Kistermacher et al., 1978; Cruse et al., 1983) as well as between monomers in solution (Langer and Golankiewicz, 1978). Additionally, the hemi-protonated cytosine base-pair in anti or syn conformation has been observed in oligonucleotides at pH 7.0 (Gray et al., 1984).

206

H \

R N- H -- -O, N ~ , ~

~ / "O---H -~ "R \H

Fig. 4. Structure of the bicytosine cation. This is the reported pairing between cytosine residues in the foldback complex of polycytidylic and polydeoxycytidylic acids.

nated. The difference in pK values between the polymer and monomer (7.4 and 4.6) indicates an energetic advantage for forming the "proton bond" in this complex (i.e. a AG = -3.82 kcal/ mole). This apparent free-energy difference may be attributed to a combination of favorable factors, including formation of an additional hydrogen bond, enhanced stacking interactions between protonated and neutral cytosine residues (Jordan and Sostman, 1973) and reduction in electrostatic free energy (Topal and Warshaw, 1976).

The stability of these "proton bonds" at physiological pH would vary as a function of the basicity of the "protonated" proton donor (i.e. as the pK of the potential proton donor cytosine increases toward physiological pH, formation of the proton bond becomes more likely). Several observations support this model. The helix-coil transition of the brominated derivative (Inman, 1964), poly-bromodeoxycytosine (BrdC), requires a higher proton concentration (lower pH, 5.5) than poly dC because of the electron withdrawing effects of bromine on the pyrimidine, which lowers the pK of 5-bromodeoxycytosine to 3.5. The bicytosine cation has also been observed in dimethyl sulfoxide (DMSO) by NMR spectroscopy (Langer and Golankiewicz, 1978). Further, the stability of bicytosine complexes for a series of 5-substituted cytosine derivatives increases with increasing basicity of the cytosine monomers; 5-ethyl (pK 4.9) > 5-methyl (pK 4.6) > cytosine (pK 4.4) (Lan- ger and Golankiewicz, 1978).

2-Aminopurine. Studies on the bicytosine cation clearly show that, in the appropriate base- pairing environment, cytosine protonation is energetically advantageous under physiological conditions. Base-pairing between cytosine and the

mutagenic base analog 2-aminopurine may also prove to be an important example of this phenom- enon. 2-Aminopurine "normally" pairs with thymine, however, the occasional pairing with cytosine is the suggested molecular basis of its mutagenic activity (Hopkins and Goodman, 1980). Al- though the mutagenicity of 2-aminopurine has been known for 30 years, the nature of base pairing between 2-aminopurine and cytosine has just been implicated in constructed polymers for the first time by nuclear magnetic resonance studies (Sowers et al., 1986a).

In the preferred conformations of the base pair (Fig. 5), only one hydrogen bond can be formed between 2-aminopurine and cytosine. Freeze (1959) first suggested the generally accepted model that this base pair may be stabilized by inversion of one of the bases into its rare imino tautomer (Fig. 5B or 5C) resulting in the formation of an additional hydrogen bond, but this structure has never been demonstrated. As in the case of the imino tautomeric structure, consideration of the potential for 2-aminopurine to exist as an ionized base pair also accommodates two hydrogen bonds which can be formed through protonation of either the purine (Fig. 5E) or pyrimidine (Fig. 5D) base in the pairing structure. All possible hydrogen- bonding schemes should be considered as being in thermodynamic equilibrium with one another as shown in Fig. 5. Recent spectroscopic evidence by Goodman and Ratliff (1983) indicated the presence of a nitrogen-nitrogen hydrogen bond between the two bases, in support of this scheme.

Both ionization and tautomerization provide an additional hydrogen bond, however, at physiological pH protonation is energetically more favored than formation of the rare tautomer. This follows from the fact that the pK values of imino tautomers are well above physiological pH so that under physiological conditions, the protonated structures should predominate. Because the cytosine residue is more basic than the 2-aminopurine residue, a proton would be found predominantly on the cytosine. Therefore, we proposed and it has recently been confirmed by nuclear magnetic resonance that structure D in Fig. 5 represents the most favorable pairing between cytosine and 2- aminopurine under physiological conditions (Sowers et al., 1986a).

A .

,, ,, ',_2__,',

H CYTOSINE 2-AMINOPURINE

B

B. R N--H ~ ~

• O---H"N

K5 ,~

g

\

K H +

D .

%?-.%y"- K3 %

1 lt(4 H +

O-..-H-N

/

Fig. 5. Possible hydrogen bonding configurations between cytosine (R = H) and 2-aminopurine.

207

(B) Base modification may induce proton transfer Formation of an ionized base pair may result

from the occasional pairing of a modified base with abnormal base during DNA replication. Base ionization may also result from modification of bases in intact DNA molecules. Under normal conditions, the proton in the nitrogen-nitrogen hydrogen bond of the guanine-cytosine base pair is located predominantly on the guanine residue because of the large difference in the basicities of the two sites (pK C, 4.5; pK G, 9.5) (Dunn and Hall, 1975). It is thermodynamically possible for the proton to tunnel to the opposing side of the base pair, thus protonating cytosine and leading to the ionization of guanine as shown in the top of Fig. 6. This very unlikely event (Kyogoku et al., 1961; Lowdin, 1965; Shulman, 1969) might greatly increase in probability if modification of guanine results in lowering the pK value associated with ionization of the N 1 proton. Such an event is supported for the interaction of the silver ion with guanine residues in DNA (Yamane and Davidson, 1962; Daune et al., 1966; Jensen and Davidson, 1966; Bloomfield et al., 1974; Zavriev et al., 1979; Dattagupta and Crothers, 1981). The accepted model for silver binding to guanine supports formation of a Type I metal ion complex with the N 7

and interaction with the 0 6 positions of the guanine base (Tu and Reinosa, 1966; Eichhorn et al., 1967). This binding results in proton transfer to the opposing cytosine residue as indicated in the lower part of Fig. 6. This ionized base-pair structure is supported by spectrophotometric and other physical data (Yamane and Davidson, 1962; Daune et al., 1966; Jensen and Davidson, 1966; Bloomfield et al., 1974; Zavriev et al., 1979; Dat- tagupta and Crothers, 1981; see refs. in Bloom- field et al., 1974 for discussion).

06-Alkylguanine. In general, the data discussed here suggests that modification of a base which results in lowering the pK value of the donor nitrogen below that of the hydrogen-bond acceptor, will induce proton transfer (charge transfer). An important extrapolation of this model involves the interaction of alkylating agents with the DNA bases. A case closely related to the silver/guanine complex is alkylation of guanine at the 0 6 position. The formation of Or-al - kylguanine is a mutagenic modification (Loveless, 1969) which appears to lead to the inducation of cancer by small alkylating agents (Pegg, 1977, for a review) and prevents incorporation of CMP into polymer by RNA polymerase (Gerchman and

208

H

/ O---H-N ~'o~O ~ N---~ ~-~N / ~H \

\ O ' - "H-N N

H d r / J , . H "~e D

R \ / ",,

N . - J \ _ . . / \ / --~O +~)-=N

"" ~H Fig. 6. Induced proton transfer between NLguanine and N3-cytosine. The top structure illustrates the equilibrium for single proton transfer between guanine and cytosine. This equilibrium may be shifted toward cytosine protonation by modification of guanine by O6-alkylation (middle structure) or silver ion binding (lower structure).

Ludlum, 1973). The pK value for the N 1 proton of guanine is 9.8. However, alkylation at the 0 6 position drops this pK value of the N 1 position to less than 2.5 (Sowers, 1983) which is below the pK of the cytosine residue (pK 4.6). We conclude that alkylation of the 0 6 position of guanine is likely to initially result in proton transfer to cytosine as shown in the middle of Fig. 6 (Sowers, 1983; Sowers et al., 1984; Richards et al., 1984).

A further equilibrium with the cytosine: O6-alkylguanine base pair involves the possible loss of the proton to solvent water. This equilibrium, however, leads to the loss of a hydrogen bond between the pairing bases. This increases the probability that cytosine may be protohated while pairing with O6-alkylguanine. Though speculative, an important impact of cytosine or 5-methylcyto-

sine protonation might be chemical destabilization leading to a higher rate of deamination of the cytosine nucleosides to uracil (or thymidine) thus leading to transition mutation. As discussed above, proton dissociation from the base pair would be resisted by formation of a hydrogen bond. There- fore, if the cytosine: O6-alkylguanine base pair resides in DNA as a result of either in s~tu alkylation or incorporation during DNA replication (Gerchman and Ludhirn, 1973), it could be stabilized by a proton bond. Although the most fre- quent cause of mutation by O6-alkylguanine appears to occur from miscoding for thymidine, the same mutant phenotype would result from destabilization of cytosine as a result of protonation leading to deamination of cytosine to uracil. In this case adenine would be the preferred pairing

base, either incorporated directly opposite uracil or opposite an AP site left by the action of uracil- DNA-glycosylase. The bonding structure for the O6G:C base pair is predicted by analogy with similar base pairing structures stabilizing both cytosine :cytosine base pairs and the Type I silver complex. Recent experimental evidence from Dr. Shaw's laboratory (Williams, Reynolds and Shaw, unpublished observations) also supports the preferred formation of ionized base-pairing structures between sialated nucleosides of.O6-alkylguanine and cytosine in orgamc soluuons conta~mng a proton source. The preferred formation of ionized base pairs between O6-alkylguanine and cytosine as well as between cytosine and cytosine is sup-

ported by NMR spectroscopy in these studies. 04-Thymine. Alkylation of the 0 4 position of

thymine (Hall and Saffhill, 1983) should also induce proton transfer to the base-paired adenine residue. The pK of the N 3 proton of thymine is 9.8, however, alkylation of the 0 4 position induces a precipitous drop in pK to below 2 (Sowers, 1983; Sowers et al., 1984; Richards et al., 1984). Proton transfer to the more basic adenine residue (pK 3.4) could then occur. However, the stability of the proton bond in the adenine : O4T base pair would be lower than that of protonated base pairs containing cytosine due to the lower basicity of adenine and greater free-energy change for adenine protonation at physiological pH.

cis- and trans-Platinum. In addition to simple alkylating agents, several other compounds of in- terest in cancer chemotherapy also bind to DNA and may effect changes in pK values, cis-Platinum coordination complexes have well established activity as anti-neoplastic agents and are used in the treatment of several human malignancies (Roberts and Thomson, 1979). Whereas the cis-platinum complexes are both mutagenic (Brouwer et al., 1981; Zwelling et al., 1979) and therapeutic, the trans-isomers do not display these biological activ- ities at equitoxic doses. Although binding properties of the two isomers with DNA are similar (Roberts and Thomson, 1979; Bradley et al., 1982), an understanding of the more subtle aspects of this interaction, which distinguish these isomers from one another, may help elucidate the neces- sary conditions for both mutagenicity and chem- otherapeutic efficacy.

209

In recent years, there has been increasing speculation that Nl-guanine deprotonation upon cis-platinum binding may have a strong bearing on the mechanism of cis-platinum anti-tumor activity (Macquet and Theophanides, 1975; Rosen- berg, 1978; Chu et al., 1978; Faggiani et al., 1980; Lippert, 1981)• The capacity of cis- and trans-isomers to influence the acidity of the guanine N 1 proton islone of the few demonstrated differences between the two isomers (Fig. 7). In aqueous solution, the pK value of the N 1 proton of guanine is only slightly affected by trans-platinum binding• On the other hand, the pK value of the N 1 proton of the cis-platinum/guanine complex is reduced by greater than 2.8 units to a pK of 7 (Chu et al., 1978; Lippert, 1981). Therefore, on the basis of influence on ionization of the N ] proton, cis-, but not trans-platinum complex formation should increase the probability of guanine mispairing. In support of this suggestion, Faggiani et al. (1980) presented crystallographic data which demonstrated an unexpected base pairing between an unmodified guanine and an N 1 deprotonated "zwitterionic" guanine/cis-platinum derivative• The base-pairing properties of both the protonated and deprotonated forms of the cis- platinum/guanine complex have been observed in DMSO (Lippert, 1981)• When protonated, the cis-platinum/guanine complex will hydrogen bond with cytosine only, however, when deprotonated it pairs with both thymine and guanine. In general, when the pK value of a potential hydrogen-bonding site is near physiological pH, half the time it acts as a proton acceptor, and half the time as a proton donor. This is the maximum situation for ambiguity in hydrogen-bond formation.

NH~ _NH3 OH ,,~P t ~ H3N'~.p( NH 3

o ~ o I--

C I S - P t TRANS- Pt

Fig. 7. Proposed structure for the guanine/cis-platinum complex which results in dissociation of the N 1 proton.

210

(C) Cytosine protonation may be coupled with guanine conformational changes

Chemical modification of DNA bases may change base-pairing characteristics directly by in- ducing dramatic alterations in the ionization of hydrogen-bonding sites. Base-pairing characteristics may also be changed by modification-induced conformational changes. The N 1 position of guanine is normally a hydrogen-bond donor (pK > 9), and the N 7 position (pK < 3), an acceptor. When guanine is in the preferred anti conformation, only the N 1 position is involved in hydrogen bonding with cytosine. Inversion of the guanine residue from the normal anti to a syn conformation results in the exchange of a hydrogen bond donor (N 1) for an acceptor (N7). In this situation, acquisition of a proton by cytosine may result in formation of an additional hydrogen bond between cytosine and a guanine residue in the syn conformation thus stabilizing the Hoogsteen base pair.

The acid structure of DNA (Zimmer and Venner, 1966; Zimmer et al., 1968; Courtois et al., 1968; O'Connor et al., 1981; Smol'janinova et al., 1982), first described by Zimmer and Venner (1966), provides an example of a protonated cytosine residue base-paired with a guanine residue in the syn conformation (Fig. 8). As the pH of a DNA solution is lowered, the most basic accessible site, N 7 of guanine, becomes protonated (O'Connor et al., 1981). Protonation of the N 7 position of guanine results in an anti to syn rotation of the protonated guanine residue as evi- denced by inversion of the CD spectrum (Courtois et al., 1968). In the resulting Hoogsteen base conformation the N 7 positions of guanine acts as a proton donor, however, because the cytosine residue (pK 4.5) is more basic than the N 7 position of guanine (pK 2.5), proton transfer to the opposing cytosine residue should occur as shown in structure IV (Smol'janinova et al., 1982).

Increase in the proton concentration (decrease in pH) provides the driving force for formation of the "acid" structure of DNA, however other modifications may drive formation of similar structures. Binding of bulky carcinogen to the C 8 (Jordan and Niv, 1977; Singer and Grinberger, 1983) and N 2 (Kadlubar, 1980) positions of guanine can also induce the anti to syn rotation.

H H ÷ R \N-H---O ..~

. . . .

P O O P

. . . . . .

HO / H

| H \N-H H I~' \ N - - ~

Fig. 8. The acid structure of DNA. As the pH of a D N A solution is lowered from 4.0 to 3.0 the N 7 position of guanine is protonated. Protonation of guanine in this position induces an anti --* syn rotation around the glycosidic bond. Subsequent hydrogen bond formation between N 3 of cytosine and N 7 of guanine results in proton transfer from guanine to cytosine.

When in the syn conformation, an additional hydrogen bond may be formed upon prot0nation of cytosine thus stabilizing base pairs containing modified guanine residues (Fig. 9). Recently, Quigley et al. (1986) presented evidence for a Hoogsteen G : C base pair where cytosine is protohated as a result of anti-syn rotation promoted by the antibiotic triostin A. In a favorable stretch of DNA sequence, a modified nucleotide like mS C can also promote formation of a left handed helix in DNA (Behe and Felsenfeld, 1981).

211

rv

/~ /~oH

/ . I . 3 / °" ~, ~ c%..~o

---H- N}4

: : ~ .. O - P

CFI3 O H

Ho 3 ~ )

Fig. 9. Mutagens can induce anti to syn rotation of guanine in DNA. This figure illustrates the conformational change induced by acetyl-AAF alkylation of guanine in DNA. As in Fig. 8, rotation of the base around the glycosidic bond could result in formation of an additional hydrogen bond between the modified guanine and a protonated cytosine residue.

(19) Base protonation and DNA stability The evidence discussed above argues that base

protonation may provide increased stability in base pairs including a modified base by facilitat- ing formation of additional hydrogen bonds. The other primary component in the stability of nucleic acids is the out of plane component of base-base interaction known as base stacking. Evidence presented from many laboratories argues that base- stacking interactions are comprised of electrostatic, polarization and dispersion components

(DeVoe and Tinoco, 1962; Broom et al., 1967; Lawaczeck and Wagner, 1974). Jordan and Sost- man (1973) calculated the effects of base protonation on intermolecular interactions and found that, in all cases, protonation of one of the bases in a stacked pair increased the magnitude of stacking interactions. In some cases, protonation can double the magnitude of the stacking interaction through introduction of a favorable monopole- induced dipole interaction (Jordan and Sostman, 1973).

Consistent with these theoretical predictions, pK values of stacked bases are generally higher than those of monomer bases in solution. The pK of dCMP in solution is 4.4, however, in single- stranded DNA, the pK of cytosine residues is increased to 4.6 (Zimmer et al., 1968). The pK of 2-aminopurine in solution is 3.8, but rises to 4.3 in polymers (Janion and Shugar, 1973).

Two additional lines of evidence demonstrate the biological importance of charged molecules in polynucleotide helices. First, base-modified nucleosides are fairly common in transfer and mes- senger RNA and these bases are generally believed to play important and specific structural roles (Rich and RajBhandary, 1976; Tazawa and Inoue, 1983). The most prevalent are the methyl- ated bases N3-CH3-C, N7-CH3-G and N1-CHa-A, all of which would predominantly carry a positive charge at physiological pH. Moreover, ionic interactions are important in the association of a number of intercalating dyes and antibiotics with DNA. These flat, aromatic molecules intercalate into the helix between base pairs. It is important to note that most intercalating agents, such as ethidium, proflavine and acridine are positively charged at physiological pH and that the charged rings are stacked within the helix (Norden and Tjerneld, 1982; Mirau et al., 1982; Saenger, 1984). These structures may be stabilizing because the positive charge within the helix can act to reduce electrostatic repulsions along the phosphate backbone (Topal and Warshaw, 1976). Dimethyl sulfate re- acts predominantly with DNA to form 7-methylguanine. Methylation of guanine at only a few positions in poly(dG-dC), poly(dG-dC) lowers relaxation time for conversion of the B form of

:DNA to the Z form. It has been suggested that conversion of B-DNA to Z-DNA (which brings

212

together the phosphate groups on opposite sides of the helix in B-DNA) may be at least partially stabilized by the positive charge on 7-methylguanine. This charge may neutralize adjoining phosphate g r o u p s - thus lowering interstrand phosphate repulsion energy associated with charge neutralization (Mrller et al., 1981; Rich et al., 1984).

(E) Hydrogen-bond formation may be a driving force for proton dissociation

In the previous sections we have argued that protonated base pairs may be a consequence of base modification. The other side of this argument is that base modification may increase proton dissociation leading to negatively charged bases. These bases could also play a role in mutagenesis. Important examples include the "zwitterionic" cis-platinum/guanine complex discussed previously and the halogenated uracil derivatives which comprise a biologically important class of base analogues. Halogen substitution on pyrimidines significantly lowers the free energy of proton dissociation at physiological pH (Fig. 10). The more electron-withdrawing the substituent, the lower the

pK value (Wempen et al., 1961; Massoulie et al., 1966) and energy associated with ionization.

Halogenated pyrimidines. Halogenated uracil derivatives presumably impart their mutagenicity by forming base pairs with guanine more fre- quently than the normal analogs, thymine and uracil (Freese, 1959; Hillebrand et al., 1984). The nature of the hydrogen bonds formed between uracil derivatives and guanine have been suggested to involve ionization (Lawley and Brookes, 1962), formation of the enol tautomer (Freese, 1959) and formation of wobble base pairs (Crick, 1966; Patel et al., 1984). The predominant form for these base pairs at physiological pH should be dependent upon their relative free energies.

In consideration of the free energy associated with base ionization and tautomerization for 5- substituted uracil derivatives (Fig. 10), it is apparent that the free energy of ionization is lower than that of tautomerization. In some cases, like that for the ionization of fluorouracil at physiological pH, the free energy for ionization is almost zero. Further, the significant difference between thymine and bromouracil is not in the tendency of

6

od

5 CI.

d

4

3

2

1

0

H

-_ 14 .H. / 4.,,/j O ~ N " pK= 8.1 1 N - H - H t

CH 3 N\ 0

/ -1.%_

CH3, / 0

61~3/N _ H I

Y pK=9.a

Fig. 10. Relative free energies of the rare tautomeric and ionized uracil derivatives. Free-energy values were derived from dissociation constants (see text).

213

these bases to tautomerize, but rather in their tendency to ionize. Based upon values determined in solution, the ionized base pair formed between uracil derivatives and guanine is more likely than pairing involving the rare tautomer. The difference in energy between the two conformational possibilities increases as the pK value of the uracil derivative decreases. Thus, in explaining the increased mutagenicity of derivatives like bromouracil, the model presented here argues for serious consideration of ionized base pairs in mutagenic base pairings. Similarly, Kulikowsky and Shugar (1978) have shown that 5-substitution of cytosine derivatives has a much more dramatic effect upon ionization than upon tautomerization. The effects on potential for hydrogen bonding of 5-substituted cytosine derivatives is illustrated schematically in Fig. 3. These relationships demonstrate the reduction in potential for hydrogen bonding caused by 5-substituted electron-withdrawing halogens and the increasing potential for cytosine ionization caused by the electron-donat- ing 5-methyl substituent (Sowers, 1983).

It is recognized that this model is in some ways incomplete. Other base-pairing structures such as wobble base pairs should also be considered, but it is not possible at this point to evaluate the energy required to form the wobble base pair. In the active site of D N A polymerase, however, the cost of base wobble may be significant based upon the apparent low frequency of G-T base-pair formation (Loeb and Kunkel, 1982).

Other factors are also important in consideration of a possible role for ionized uracil derivatives in base pairing. Introduction of a positive charge into the DNA helix is thermodynamically advantageous, however, the influence of a negatively charged base is not known. In this regard, it is important to point out that the negative charge associated with base ionization is distributed over the molecule which distinguishes it from the highly localized charge density on a group such as phosphate. Further, in the case of bromouracil, one would have to provide evidence that the negative charge in the helix had a destabilizing influence of a least 3 kcal/mole in order to support an argument for a tautomeric over an ionized base as a preferred base-pairing partner. (This represents the difference in energy between the ionized base and the rare tautomer at physiological pH).

(F) Tautomers in DNA This paper has focused upon the formation of

ionized base pairs and its possible significance in mutagenesis. For the modified bases discussed above, the ionized base pair is lower in energy than the rare tautomer. The model presented here, however, is consistent with evidence which indicates that tautomerization may be a consequence of some other base modifications. For example, replacement of a hydrogen atom on the exocylic amino group of cytosine with an electron- withdrawing substituent shifts the conformational equilibrium toward the rare tautomer (Singer and Kusmierek for a review, 1982). The free energy associated with tautomerization of the modified cytosine derivatives fails from 6 kcal/mole for cytosine (Fresco et al., 1980) to near zero with the 4-hydroxy and 4-amino substituents (Singer and Kusmierek, 1982). N4-Aminocytosine, the reaction product formed between cytosine and hy-

, drazine, will exist predominantly in the imino tautomeric conformation which can pair with adenine as seen in in vitro incorporation studies (Brown and Hewlins, 1968; Brown et al., 1968; Singer and Kusmierek, 1982).

IV. Implications of the model

In this discussion, we have attempted to illustrate interrelationships among a wide range of mutagenic base analogues. We have argued that many base pairs involving important promutagenic base analogues show an increased tendency to ionize relative to normal conformers under physiological conditions (see Fig. 3). Conse- quences of ionized base-pair formation may include perturbation of DNA conformation, accelerated base hydrolysis, and improper base selection during DNA replication resulting in base substitution mutations.

Recent studies have shown that DNA conformation is dynamic and that many conformations are in thermodynamic equilibrium with each other (Sowers, 1983; Sowers et al., 1986a, b). Base modification [including N7G alkylation which intro- duces a charge into the helix (MSller et al., 1981)], salt concentration (Pohi and Jovin, 1972), base protonation (Chela, 1984) a n d protein binding (Rich et al., 1984) all can significantly shift DNA

214

conformational equilibria away from B form DNA and/or cause anti to syn rotation of base pairs. Syn conformers of bases also are more susceptible to acid hydrolysis (Jordan and Niv, 1977) illustrative of the direct link between DNA conformation, stability and reactivity. Structural perturbations, which result from base ionization, may be advantageous in providing recognition signals for repair enzyme (Pulleyblank et al., 1985). On the other hand, such perturbations may be deleterious through disruption of DNA interactions that are dependent on specific polynucleotide conformations.

As mentioned above, however, DNA conformation is only one of the aspects of DNA structure that may be affected by ionized bases. The base composition of DNA may also be directly altered, because although ionized bases may be thermody- namicaUy stable under physiological conditions, they may be kineticaUy unstable. Base hydrolysis reactions, including deamination (Lindahl and Nyberg, 1974) of cytosine and adenine derivatives and depurination (Lindahl and Nyberg, 1972) of guanine derivatives are all significantly accelerated by base protonation.

We have presented evidence that many ionized base pairs are thermodynamically stable in DNA under physiological conditions. Moreover, this evidence suggests that ionized base pairs may stabilize interactions in the active site(s) of DNA polymerases during DNA replication. Interactions between bases, including both base stacking and hydrogen bonding, have significant electrostatic and polarization components and the magnitude of these interactions will increase with a decrease in the dielectric constant of the solvent environment. Several of the proposed ionized base pairs discussed here, including the bicytosine cation and guanine/cis-platinum complexes, have been observed in organic solvents which have lower dielectric constants than water [e for water, 78; DMSO, 47; chloroform, 4; (Riddick and Bunger, 1970)]. As discussed previously, evidence from NMR studies of nucleoside analogues in organic solvents (Williams, Reynolds and Ramsay-Shaw, unpublished observations) indicates that hydrogen bonding between Or-alkylated guanosine and cytidine can be observed only when one of the bases is ionized. Further, the magnitude of the

interactions calculated in the vacuum (e = 1) between a neutral and an ionized base are generally greater than those between neutral bases (Jordan and Sostman, 1973). Therefore, if the dielectric constants of the active sites of enzymes like DNA polymerase are lower than water, the magnitude of interactions between neutral and ionized bases may be even higher than predicted from studies in aqueous solution (Petruske et al., 1986).

Since ionized base pairs may play a role in base selection by DNA polymerases, special attention should be paid to the role of solvent pH when comparing the properties of different DNA polymerases. As discussed above, the stability of ionized base pairs is highly pH dependent. Since DNA polymerases have clear pH optima (e.g. pol a has a pH optimum of 7.2 whereas the pH optimum for polymerase fl is 8.5) the stability of ionized base pairs may be especially relevant in studies of the fidelity of DNA polymerases. The model presented here suggests that a standard pH must be chosen for comparative study of the effects of base analogues on DNA polymerase fidelity.

In development of this model, we have em- phasized a thermodynamic approach to analysis of mutagenic base-pairing interactions. As most genetic mutations are believed to result from aberrant hydrogen-bond formation, we have presented a thermodynamic framework for prediction of the most likely base-pairing configuration accruing from the equilibrium between the normal, ionic and rare tautomeric forms of the DNA bases (see Fig. 1). Significantly, the model argues that in many cases ionized forms are lower in energy than rare tautomers and that in these cases tautomers may be of lesser biological relevance than previously believed. In particular, thermodynamic evidence, as well as evidence from work with model polymers, suggest that ionized base pairs may be relatively common in DNA-containing modified bases. Since analysis of damage and mutation is now possible at the DNA-sequence level, events occurring at and in the region of a specific modified base have become accessible for mechanistic considerations. In this context, we suggest that ionized bases in DNA could have important ramifications for chemical reactivity of both modified bases and their cross-strand pairing partners,

as well as for DNA structure of the base pairs immediately surrounding the modified base.

Acknowledgement

This work was supported by an NCI Research Grant No. RO1CA31110 (WDS). Support for this work also came from Grant GM-23681 (BRS).

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DNA base modification: Ionized base pairs and mutagenesis