Mutation Research, 214 (1989) 13-22 13 Elsevier

MTR 02553

Oxidized apurinic/apyrimidinic sites formed in D N A by oxidative mutagens

Lawrence F. Povirk and Robert J. Steighner Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University,

Richmond, VA 23298 (U.S.A.)

(Received 9 May 1988) (Accepted 22 February 1989)

Keywords: Bleomycin; Neocarzinostatin; "y-Radiation; Apurinic/apyrimidinic sites in DNA

Summary

Treatment of DNA with any of several agents, including ionizing radiation, hydrogen peroxide, bleomycin, neocarzinostatin and the copper (I) chelate complex of 1,10-phenanthroline, produces apurinic/apyrimidinic (AP) sites containing oxidized deoxyribose moieties. These AP sites, which are formed by specific or nonspecific free-radical attack on deoxyribose, have been shown to involve oxidation of deoxyribose at the C-1', C-2' or C-4' position. Oxidized AP sites are generally more susceptible to chemical cleavage than normal AP sites, but are in some cases resistant to cleavage by repair AP endonucleases. Nearly all of the AP sites produced by neocarzinostatin, and a fraction of those produced by bleomycin, are accompanied by closely opposed breaks in the complementary strand. Sequence specificity data strongly implicate oxidized AP sites in neocarzinostatin-induced mutagenesis. The role of AP sites in mutagenesis by the other oxidative mutagens is less clear, although there is in some cases suggestive evidence for such a role.

Free radicals have been shown to induce a multitude of different DNA lesions (reviewed by Hutchinson, 1985, and by von Sonntag, 1987), and the biological effects of individual lesions have been difficult to assess. One class of such damage consists of various forms of oxidized apurinic/ apyrimidinic (AP) sites, resulting from free radi- cal-induced oxidation of DNA sugars. Agents which induce specific types of DNA sugar damage, including oxidized AP sites, have allowed de- termination of some of the properties and possible biological effects of these lesions.

Correspondence: Dr. Lawrence F. Povirk, P.O. Box 230. Medical College of Virginia, Richmond, VA 23298 (U.S.A).

Formation and structure

The formation of apurinic/apyrimidinic (AP) sites in DNA may occur spontaneously at physio- logical pH, temperature and ionic strength (Lin- dahl and Nyberg, 1972). The rate of base release increases with increasing temperature and decreas- ing pH with purine bases being released at a higher rate then pyrimidines (Kochetkov and Budovskii, 1972). Chemical modification of the purine or pyrimidine ring by alkylating agents may also decrease the stability of the N-glycosylic bond, leading to base release (Tarpley et al., 1982). Hydrolysis of the N-glycosylic bond may also occur enzymatically in response to the actions of DNA glycosylase enzymes (Duncan, 1981). The

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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CH2 CH z

0 0 ® ®

i i

i i

IA IB

I CH2

IC

® ® ® 0 0 0

I I I CH 2 CH2 CH2

H o - ~ O H ~ 0 = ~ = 0 - - H O - ~ OH

O O ® ®

i

I 2A 2B ?.C

® ® 0 0 I i CH 2 CH2 CH2

- ° :oL 0 0 ® ®

l

1 5A 5B 4

Fig. 1. Structures of AP sites and some of their #-elimination products. (1) Normal AP site resulting from simple hydrolysis of the N-glycosyl bond. (2) Bleomycin-induced AP site, involv- ing oxidation of C-4'. (3) AP site, involving C-I' oxidation, induced by -/-rays, (OP)2Cu +, and hydrogen peroxide. (4) Another AP site induced by -/-rays, involving C-2' oxidation.

resultant deoxyribose residue in DNA exists in equilibrium between the furanose and free al- dehyde forms (structures 1A and 1B in Fig. 1) (Overend, 1950). Subsequent alkali treatment re- sults in strand scission by a fl-elimination reaction (Kochetkov and Budovskii, 1972). .-

In addition to these AP sites (hereafter referred to as normal AP sites), several types of modified AP sites have been identified in DNA treated with various agents which form free radicals (Fig. 1). These AP sites are believed to result from either specific or nonspecific hydrogen abstraction from

the C-1', C-2' or C-4' positions of deoxyribose, resulting in an oxidation of the sugar moiety which releases the DNA base without cleavage of the phosphodiester backbone.

Treatment of DNA with bleomycin. Fe 2+ plus oxygen results in the formation of two distinct lesions, both of which may arise from the abstrac- tion of a hydrogen atom from the C-4' position of the deoxyribose group. Anaerobically treated DNA preferentially releases an unmodified base leaving an intact DNA chain containing an AP site which is susceptible to alkali-mediated cleavage (Burger et al., 1982). Aerobically treated DNA contains roughly equal amounts of AP sites and single- strand breaks. The single-strand breaks formed in bleomycin-treated DNA are associated with the release of a base propenal, leaving 5-phos- phomonoesterase and 3'-phosphoglycolate termini (Giloni et al., 1982). Recent studies of the alkali- labile AP site have revealed a site different from that induced by mild acid hydrolysis.

Sugiyama et al. (1985) treated a self-comple- mentary dodecamer with bleomycin and analyzed the products by HPLC. Knowledge of the se- quence specificity of bleomycin allowed the authors to hypothesize which products may be formed upon subsequent alkaline treatment of the DNA. Chemical synthesis of the expected prod- ucts and analysis of their retention times resulted in the identification of a dinucleotide-2,4-dihy- droxypentenone derivative in the alkali-treated sample. This product is expected to arise from a 4'-hydroxylated deoxyribose group (structure 2A) via fl-elimination (2C), followed by rearrangement to form a 5-carbon pentenone ring. Chemical synthesis of the expected fl-elimination product (2C), followed by alkaline treatment, produced the same pentenone structure, thus confirming the proposed rearrangement reaction.

Subsequent work by Rabow et al. (1986) led to the direct quantitation of the modified deoxyri- bose group. Rabow et al. incubated a double stranded hexamer with bleomycin under anaerobic conditions, separated the products by reverse phase HPLC and quantitated the yield of free cytosine. Correlation of the amount of free cytosine re- leased with the appearance of an unknown major peak on HPLC allowed the authors to surmise that the unknown peak consisted of the AP site-

containing hexamer. Reduction of the hexamer with NaB3H4 trapped the alkali-labile deoxyribose species in a chemically stable form. Subsequent enzymatic digestion with P1 nuclease, alkaline phosphatase and snake venom phosphodiesterase yielded a carbohydrate moiety indistinguishable chromatographically from authentic 2-deoxypenti- tol. The formation of this derivative is consistent with an AP site involving hydroxylation (subse- quent to hydrogen abstraction) at the C-4' posi- tion of deoxyribose (structure 2A), in DNA treated with bleomycin. The demonstrated presence of both the erythro and threo deoxypentitol isomers implies the formation of a ketone at C-4' (struc- ture 2B), and its subsequent reduction. Thus, this type of AP site, like the normal AP site, probably exists in equilibrium between ring-closed and ring-opened forms. 3H release studies, using sub- strates specifically labeled at various positions in the deoxyribose ring, were also consistent with this structure (Wu et al., 1983).

von Sonntag (1987) and coworkers have exten- sively investigated DNA damage induced by y- radiation, which is mediated primarily by hy- droxyl radicals. Although most hydroxyl radicals add across the double bonds of the bases, roughly 20% may be involved in hydrogen atom abstrac- tion from deoxyribose (Scholes et al., 1960). In addition to the generation of normal AP sites resulting from N-glycosylic bond labilization sub- sequent to base modification, two modified AP sites have been shown to occur in irradiated DNA.

Dizdaroglu et al. (1977a) detected the forma- tion of 2-deoxyribose-o-erythro-pentonic acid ( C H E O H - C H O H - C H O H - C H 2-COOH ) from calf-thymus DNA irradiated under both aerobic and anaerobic conditions. In these studies, the y-irradiated DNA was subjected to vigorous al- kaline treatment to release the AP sugars from DNA, followed by treatment with alkaline phos- phatase to remove phosphate groups. Subsequent trimethylsilylation followed by gas chromatogra- phy and mass spectral analysis resulted in the identification of the pentonic acid derivative of deoxyribose. This species is believed to arise by the abstraction of hydrogen from the C-1' posi- tion, followed by base release and the formation of the alkali-labile AP site with a lactone at C-1', i.e. 2-deoxy-o-erythro-pentonolactone (structure

15

3A). The lactone presumably hydrolyses to yield the observed ring-opened sugar with a carboxylic acid at C-I'. The lactone-type AP site has also been invoked to explain the strand breaks with 3'- and 5'-phosphate termini induced by the Cu r chelate complex of 1,10-phenanthroline [(OP)2 Cu r ] in the presence of hydrogen peroxide (Kuwabara et al., 1986), as well as by hydrogen peroxide alone (Rhease and Freese, 1967). The (OP)2Cu÷-induced breaks are sequence-specific (Spassky and Sigman, 1985), suggesting that they arise by site-specific attack on deoxyribose by DNA-bound (OP)2Cu 4. Goyne and Sigman (1987) showed that, when the (OP)2Cu÷-treated DNA was heated to eliminate labile intermediates, the deoxyribose derivative 5-methylene-2-5H-fura- none (structure 3B), identified by mass spec- trometry, was released. The formation of this species was stoichiometric with the formation of breaks with 3'-phosphate termini. Thus, it was proposed that the breaks arose from the lactone- type AP site (structure 3A) by fl-elimination/~- elimination (giving 3B), although the presence of intact AP sites in the treated DNA was not specifically demonstrated. Rhease and Freese (1967) invoked a similar mechanism to explain hydrogen peroxide- induced alkali-dependent breaks in oligo (dA). However, they proposed ring-opening of the lactone to form the pentonic acid prior to/3-elimination, based on the observa- tion that peroxide treatment of free adenosine produced the pentonic acid derivative.

Further studies by Dizdaroglu et al. (1977b) utilized NaBD 4 to trap carbonyl-containing de- oxyribose derivatives in y-irradiated DNA prior to their release by alkaline treatment. Using the same methods employed to quantitate the pentonic acid derivative, the authors measured the formation of meso-erythritol-l-dl ( C H E O H - C H O H - C H O H - CDOH). This species is thought to arise from hydrogen abstraction at the C-2' position fol- lowed by the r-fragmentation of an oxyl radical intermediate, resulting in the loss of the base and attached C-1' carbon atom from the deoxyribose residue. The residual AP site consists of an eryth- rose group with an aldehyde at C-2' (structure 4), which is reduced to the alkali-stable erythritol prior to isolation.

Using similar methods, Isildar et al. (1981)

16

subsequently confirmed the formation of the lac- tone-type AP site (structure 3A), by isolating and identifying its expected NaBD 4 reduction product, 2-deoxy-erythro-pent i tol- l -d 2 (CH2OH-CHOH- C H O H - C H 2 - C D 2 O H ). Thus, two types of oxidized AP sites (structures 3A and 4) have been identified in "/-irradiated DNA. Sugar residues similar to the bleomycin-induced AP site have also been detected, but it is unclear whether these are present in DNA with both phosphodiester bonds still intact (Isildar et al., 1981).

A number of studies have shown that neocarzi- nostatin (NCS), particularly when activated by glutathione cofactor, induces AP endonuclease- susceptible sites with chemical properties distinct from those of normal AP sites (Bose et al., 1980; Kappen and Goldberg, 1983; Povirk and Gold- berg, 1985). Formation of these putative AP sites is oxygen-dependent (Kappen et al., 1984), sug- gesting an oxidation of deoxyribose. Recent stud- ies in which these lesions were formed in synthetic oligonucleotides have clearly demonstrated the ex- istence of an oligonucleotide from which a base has been released without cleavage of the sugar- phosphate backbone (Kappen et al., 1988). Thus, there is little doubt that the lesions are in fact AP sites. Studies with the same oligonucleotide showed that hydrogen was abstracted from the 1' and /o r 2' positions of the nucleotide at which the AP site was formed. Thus, it seems likely that these le- sions involve oxidation of the C-I ' and /o r C-2' positions (most probably C-I'), as in structures 3A and 4. However, the exact chemical structure of these AP sites remains to be determined.

The NCS-induced AP sites have the unique property that they are virtually always accompa- nied by a nearby ("closely opposed") break in the complementary strand (Povirk and Houlgrave, 1988a). This finding, along with recent proposals of a biradical form of activated NCS (Myers, 1987), suggests that this drug simultaneously at- tacks two sugars in opposite strands of DNA. A minor fraction (7-10%) of bleomycin-induced AP sites are also accompanied by a closely opposed break in the complementary strand (Povirk and Houlgrave, 1988a).

Chemical cleavage

In general, oxidized AP sites are more labile than normal AP sites, which are reasonably stable

in double-stranded DNA at neutral pH. Lafleur et al. (1981) showed that alkali-labile sites in ~,-irradiated DNA, presumably oxidized AP sites, were cleaved about 20-40-fold more rapidly than normal AP sites under both neutral and alkaline conditions in both single-stranded and double- stranded DNA; for example, the half-fife of a radiation-induced alkali-labile site in single- stranded DNA at pH 6.9 and 37°C was ap- proximately 20 rain, as opposed to 10 h for nor- mal AP sites. In all cases, rates of cleavage were 3-10-fold slower in double-stranded than in single-stranded DNA. Curiously, although ,{-rays induce at least two types of oxidized AP sites, cleavage of irradiated DNA by either heat or alkali followed a single exponential. This result suggests that either both types of sites have similar labilities, or one type of site predominates; the proportions in which the two types of sites are formed have not been determined. NCS-induced AP sites are likewise more labile than normal AP sites at all pH values; at pH 9.5, complete clea- vage of NCS-induced AP sites can be achieved under conditions where cleavage of normal AP sites is undetectable (Kappen et al., 1988). Al- kaline cleavage of bleomycin-induced AP sites is also more rapid than that reported for normal AP sites (Lloyd et al., 1978).

Sequencing gel analysis of single-stranded DNA fragments generated by AP site cleavage has re- vealed that the 3' and 5' phosphodiester linkages are differentially labile. Cleavage of normal AP sites by heating at neutral pH or treatment with mild alkali releases only the 3' phosphate, leaving the AP sugar attached via its 5' carbon to the 3' end of DNA. As has been shown by Bailley and Verly (1987), this cleavage is not a phosphodiester hydrolysis, but a fl-elimination, which results in formation of a (2 ' -3 ' ) double bond in the AP sugar (structure 1C). Vigorous alkaline treatment is required to release this sugar derivative from the 3' end of DNA. Putative AP sites induced by (OP)2Cu ÷ likewise form a break with an oxidized AP sugar (presumably the fl-elimination product of structure 3A) attached to the 3' end of D N A . The AP sugar can be released by vigorous alkaline treatment (Kuwabara et al., 1986). In the case of the NCS-induced AP site, however, sequencing gels indicate the presence of phosphate groups on both termini of the break, even when the site is

cleaved enzymatically and the DNA is not ex- posed to alkali. Thus, both the 3' and 5' linkages of the NCS-induced sites are more labile than in normal AP sites (Kappen et al., 1988). Sugiyama et al. (1985) have shown that alkaline hydrolysis of the bleomycin-induced AP site yields a break with a dihydroxycyclopentenone group (derived via a rearrangement of the AP sugar) at the 3'-end of DNA. This type of break may account for the minor bands seen upon electrophoresis of bleomycin-treated DNA, which have a slower mo- bility than the bands corresponding the direct strand breaks with 3'-phosphoglycolate termini (D'Andrea and Haseltine, 1978).

Normal AP sites can be converted to an alkali- resistant form by sodium borohydride reduction of the C-I' aldehyde (Hadi and Goldthwait, 1971). However, while the work of Dizdaroglu et al. (1977b), Isildar et al. (1981) and of Rabow et al. (1987) indicates that the various ,/-ray-induced sites and the bleomycin-induced site can be re- duced with borohydride, reversal of alkaline labil- ity has not been demonstrated. In fact, NCS-in- duced AP sites have been shown to remain alkali- labile following borohydride treatments which were sufficient to stabilize normal AP sites (Povirk and Goldberg, 1985; Kappen et al., 1988). Inter- pretation of reduction data is complicated by the wide variations in conditions used for both reduc- tion and subsequent alkaline hydrolysis; for exam- ple, sufficiently strong alkaline treatments will cleave even borohydride-reduced normal AP sites, presumably via a cyclic phosphate intermediate (Isildar et al., 1981).

Certain primary amines also catalyze cleavage of AP sites. Of several amines examined by Lin- dahl and Andersson, putrescine (10 mM) was most effective, increasing the rate of spontaneous AP site hydrolysis 30-fold at 70 o C. Recently, Povirk and Houlgrave (1988a) showed that bleomycin-in- duced and NCS-induced AP sites were efficiently cleaved at 37°C by 5-10 mM putrescine, while normal AP sites were cleaved only very slowly at this temperature even at high putrescine con- centrations (> 20 mM). Curiously, spermidine ef- ficiently cleaved normal AP sites at 37 o C, but was much less effective in cleaving bleomycin-induced AP sites. NCS-induced sites were cleaved by putrescine, spermidine and to some extent even by

17

tris(hydroxymethyl)aminomethane buffer. The reasons for these differences in susceptibility to primary amines are not known. However, the sensitivity of oxidized AP sites to cleavage by primary amines raises the possibility that they may rapidly be cleaved in cells by polyamines or by lysine amine groups of DNA-bound histones; such cleavage could profoundly affect the biologi- cal consequences of these lesions.

Normal AP sites have also been shown to form DNA interstrand crosslinks (Freese and Cashel, 1964) and DNA-protein crosslinks (Mirzabekov et al., 1978), probably through Schiff's base lin- kages between the C-I' aldehyde and primary amine groups of DNA bases or histones. To our knowledge, no attempts have been made to de- termine whether oxidized AP sites form similar crosslinks.

Susceptibility to AP endonucleases

AP sites are normally repaired by an excision pathway, beginning with cleavage of the DNA strand at the AP site by either of two types of AP endonucleases (reviewed by Lindahl, 1982). Type I enzymes cleave at the 3' side of the AP site leaving a 5'-phosphate terminus and an AP sugar attached to the 3' terminus. Type II enzymes cleave at the 5' side of the AP site, leaving a 3'-hydroxyl terminus and a 5'-phosphoryl AP sugar attached to the 5' terminus. Oxidized AP sites represent modified substrates which may not be recognized by these enzymes.

Bleomycin-induced AP sites, involving C-4' oxidation (structure 2A), were found to be as sensitive as normal AP sites to cleavage by either of two type I endonucleases, E. coli endonuclease III and the pyrimidine dimer glycosylase/AP en- donuclease of M. luteus (Povirk and Houlgrave, 1988a). These results are expected, since both nor- mal and bleomycin-induced sites can exist in a ring-opened form with a C-1' aldehyde, and type I endonucleases are believed to function by catalyz- ing fl-elimination from the C-1' aldehyde (Bailly and Verly, 1987). However, bleomycin-induced AP sites are relatively resistant to E. coli exonuclease III, which has type II AP endonuclease activity (Niwa and Moses, 1981); the bleomycin-induced sites require about thirtyfold more enzyme than

18

normal AP sites (Povirk and Houlgrave, 1988a). This result is rather surprising, since type II endo- nucleases are believed to recognize stereochem- ically the absence of the DNA base (Takeshita et al., 1987); in fact, exonuclease III even cleaves sites in which the DNA base is replaced by urea (Kow and Wallace, 1985). On the other hand, bleomycin-induced AP sites show no resistance to rat-liver AP endonuclease (Schyns e ta ! . , 1978) and only slight resistance to E. coli endonuclease 1V (Povirk and Houlgrave, 1988a); both these enzymes are also type II endonucleases. The small fraction of bleomycin-induced AP sites that are accompanied by a closely opposed strand break are resistant to all AP endonucleases, although some cleavage can be seen with very high con- centrations of endonuclease IlI.

NCS-induced AP sites show slight resistance to endonuclease IV, and profound resistance to en- donuclease III and exonuclease III; nevertheless, some cleavage has been seen with all 3 enzymes at sufficiently high concentrations (Povirk and Houlgrave, 1988a). As virtually all NCS-induced AP sites are accompanied by closely opposed breaks in the complementary strand, resistance to enzymatic cleavage may be due to the presence of the break, rather than to the chemistry of the AP site itself. It is interesting that the closely opposed AP site/strand break lesions induced by NCS are more susceptible to cleavage by endonuclease IV than similar lesions induced by bleomycin, even though nonopposed bleomycin-induced sites are efficiently cleaved. This difference could be due to the fact that the bleomycin-induced strand break involves loss of a base, while the NCS-induced break does not (Giloni et al., 1981; Kappen and Goldberg, 1983); thus, the NCS-induced sites might more easily adopt a configuration similar to double-stranded DNA.

Wallace (1983) quantified various endonucle- ase-susceptible sites in X-irradiated supercoiled DNA, and concluded that X-ray-induced oxidized AP sites were susceptible to yeast endonuclease E (a type II AP endonuclease) but not to E. coli endonuclease III. Although lesions susceptible to cleavage by endonuclease III were detected, it was concluded that these were not AP sites, but rather sites of oxidized bases, which would be cleaved by endonuclease III through a combination of its

glycosylase and AP endonuclease activities. Since the effects of alkali and endonuclease III were approximately additive, there were apparently few sites with the properties of the bleomycin-type AP site, i.e., susceptible to both alkali and endo- nuclease III. This result agrees with the proposal (see above) that most AP sites induced by ionizing radiation are associated with C-1' or C-2' oxida- tion. Since these types of sites cannot form C-1' aldehydes, their resistance to type I AP endo- nucleases such as endonuclease III is not surpris- ing.

Role in mutagenesis

There is considerable evidence that normal AP sites, at least under some circumstances, are highly mutagenic (reviewed by Loeb and Preston, 1986). The most direct evidence has come from studies in which single-stranded phage DNA is heat-de- purinated in vitro, and transfected into SOS-in- duced E. coli. The resulting mutations are pre- dominantly single-base substitutions at sites of purines in the phage DNA. The specificity of the base substitutions is consistent with preferential insertion by DNA polymerase of adenine residues, and to a lesser extent thymine and guanine re- sidues, opposite putative AP sites in the template. The mutations are completely dependent on SOS functions (i.e., the u m u D C and recA gene prod- ucts), and can be eliminated by cleavage of AP sites in the DNA with an AP endonuclease or with alkali, prior to transfection. Mutagenesis of SV40 (Gentil et al., 1984) and pBR322 (Bichara and Fuchs, 1985) by in vitro heat-depurination of dou- ble-stranded DNA, has also been reported. Foster and Davis (1987) have obtained genetic evidence that AP sites are intermediates in some of the mutations induced in E. coli by N-methyl-N'- nitro-N-nitrosoguanidine.

Since even nonspecific free-radical attack on DNA, e.g, by X-rays, induces a significant number of oxidized AP sites, these lesions are logical candidates for intermediates in mutagenesis by any agent which induces free radicals. However, in most cases it is difficult to demonstrate the in- volvement of oxidized AP sites, since numerous other potentially mutagenic lesions, such as vari- ous oxidized bases, are also formed. For example,

Tindall et al. (1988) sequenced a number of cI- mutants induced by T-irradiation of lambda phage. Two distinct classes of mutations were seen: SOS- independent mutations, primarily G : C ~ A : T transitions, and SOS-dependent mutations, which included a variety of base substitutions in which the "new" base pair was virtually always A:T. It was postulated that these mutations could have resulted from selective incorporation of adenine residues during SOS-dependent bypass of any of several "noncoding" lesions, including oxidized AP sites and damaged bases.

Since DNA damage induced by NCS and bleomycin is restricted to the sugar moiety, these agents offer an opportunity to assess the role of sugar damage, particularly oxidized AP sites, in mutagenesis. A correlation has been established between the sequence specificity of NCS-induced AP sites, and that of a specific class of NCS-in- duced mutations, namely G : C ~ A : T transitions (Povirk and Goldberg, 1985, 1986). For example, cytosines in the sequence A - G - C are hotspots both for formation of AP sites, and for G : C A : T transitions, in both the cI gene of lambda phage and the lacI gene of E. coli; furthermore, sequences where AP sites are never formed, such as G-G-C , C - G - C and G-C-C, appear to be hypomutable. Thus, NCS-induced AP sites are strongly implicated in the generation of NCS-in- duced G : C ~ A : T transitions. The role of AP sites in NCS-induced mutagenesis at A : T base pairs is less clear. However, we have recently detected, in NCS-treated lacI DNA, AP sites at certain thymine residues, including those corre- sponding to the two most frequent sites of NCS- induced A : T ~ T : A transversions (L.F. Povirk, unpublished). Thus, although the data are not yet conclusive, it appears likely that, in E. coli, oxidized AP sites may account for the majority NCS-induced base substitutions at both G : C and A : T base pairs.

The fact that NCS-induced oxidized AP sites are accompanied by closely opposed strand breaks may contribute substantially to their potent muta- genicity. As discussed above, the presence of the break apparently confers resistance to AP-endo- nuclease-mediated repair, thus increasing the probability that the replication fork will reach the AP site before it is repaired. Furthermore, at-

19

tempts by the cell to repair the strand break may involve repair synthesis across the AP site in the opposite strand, so that mutagenesis (i.e., the change in DNA sequence) may occur before nor- mal replicative DNA synthesis.

In the case of bleomycin, there is also a correla- tion in sequence specificity between mutations and AP sites (Povirk, 1987); sequenced bleomy- cin-induced mutations in lambda phage occur primarily at pyrimidines in G-C, G - T and A-T sequences, the expected positions of AP sites (as well as strand breaks). The mutations are SOS-de- pendent, as expected for AP site-mediated muta- genesis (Povirk and Houlgrave, 1988b). However, other experiments have failed to support a role for AP sites in bleomycin-induced mutagenesis. For example, host-cell defects in repair AP en- donucleases were found to have no effect on the mutation frequency of bleomycin-damaged lambda phage (Povirk and Houlgrave, 1988b). Further- more, preliminary results indicate that cleavage of bleomycin-induced AP sites in lambda DNA by endonuclease IV in vitro (followed by repackaging to form infective phage) also has no effect on mutation frequency. However, treatment of the DNA with putrescine produces a substantial (2-4-fold) decrease in mutation frequency (L.F. Povirk and R.J. Steighner, unpublished). This re- sult raises the possibility that only the bleomycin- induced AP sites accompanied by closely opposed breaks (which can be cleaved by putrescine but not by endonuclease IV) are significantly muta- genic, perhaps for the same reasons discussed in the case of the NCS-induced AP sites.

Thus, while the results with bleomycin and NCS suggest that oxidized AP sites, like normal AP sites, can serve as substrates for SOS-depen- dent bypass replication, they also suggest that lone oxidized AP sites (without a closely opposed break) may have a low mutagenic potential. This is an important issue, since nonspecific free radical damage to DNA, e.g., by X-rays, would be ex- pected to produce only a very small fraction of sites with closely opposed damage in the comple- mentary strand.

Recent studies by Loeb et al. (1988) have at- tempted to assess the possible role of AP sites in mutagenesis resulting from nonspecific free radi- cal-induced DNA damage. Treatment of single-

20

stranded ~X174 DNA with Fe z+, under condi- tions expected to generate hydroxyl radicals, was found to efficiently induce reversions of the a m3

mutation, which can only be reverted by a base substitution at a specific adenine residue in the phage DNA. Revertants were only seen when the DNA was transfected into SOS-induced (ultra- violet-irradiated) host cells, and among the re- vertants, A ~ T transversions were predominant, with a few A ~ C transversions. These results suggest that mutagenesis could be due to SOS-de- pendent replication across some "noncoding" le- sion (such as an AP site) in the phage DNA template, with preferential insertion of an adenine residue opposite the lesion. However, treatment of the DNA with alkali or with a human AP endo- nuclease reduced mutagenesis only slightly (17- 46%), suggesting that mutagenesis resulted prim- arily from alkali-stable lesions, i.e., lesions other than AP sites, possibly damaged adenine bases. Nevertheless, the partial reversal of mutagenesis by alkali may represent the loss of a component of mutagenesis mediated by oxidized AP sites. More- over, these experiments could have underesti- mated the mutagenic potential of oxidized AP sites, since a large proportion of these labile sites may have been eliminated by spontaneous clea- vage during in vitro DNA manipulations, even in samples not treated with alkali.

Conclusions

While considerable progress has been made in defining the structure and chemical properties of oxidized AP sites, the biological effects of these lesions are still in doubt. Mutagenesis studies with the radiomimetic agents bleomycin and neocarzi- nostatin suggest that, under certain conditions, oxidized AP sites can be highly mutagenic, par- ticularly when there is also closely opposed damage in the complementary strand. In the case of less specific oxidative mutagens, the SOS-dependency and base specificity of mutagenesis make oxidized AP sites prime candidates for mutagenic lesions, but in most cases other types of oxidative D N A damage could equally well be invoked. As the chemical and biochemical properties of oxidized AP sites become more clearly defined, methods may yet be developed for assessing their particular

role in mediating the genotoxic effects of oxidative mutagens.

Acknowledgements

We thank Dr. I.H. Goldberg and Dr. L.A. Loeb for helpful discussions and for communi- cating their results prior to publication. The pre- paration of this review was supported by Grant CA40615 from the United States Public Health Service, DHHS.

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