Influence of nucleotide excision repair of Escherichia coli on radiation-induced mutagenesis of double-stranded M13 DNA.

To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).

Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA.

Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.

Mutations induced by gamma-irradiation of M13 bacteriophages containing single-stranded DNA.

Oxygenated suspensions of M13 bacteriophages, containing single-stranded M13mp10 DNA, were gamma-irradiated followed by infection of E. coli cells. Mutants in the mutational target sequence, which consists of the lac promoter /operator region, the lacZ alpha gene, and a 144 bp inframe insert in the lacZ alpha gene, were selected and characterized. Except for three one-base deletions, all of the 51 mutations characterized were base substitutions. All base substitutions appeared to involve guanines and cytosines and none affect adenines and thymines. Since most of the known repair systems do not act on single-stranded DNA, the conclusion can be drawn that radiation induces under these conditions only mutagenic damages on guanine and cytosine. Although all possible G- and C-transversions and transitions were found, there is a strong preference for G-->C and G-->T transversions (21 and 25% of all base substitutions, respectively) and C-->T transitions (48% of all base substitutions). These results indicate, that the G/C-->C/G and G/C-->T/A transversions, found after irradiation of double-stranded M13 DNA, are mainly due to radiation guanine products, whereas cytosine damage is mainly responsible for G/C-->A/T transitions.

Influence of the antioxidant N-acetylcysteine and its metabolites on damage induced by bleomycin in PM2 bacteriophage DNA.

Bleomycin is considered to be a useful model compound for studying environmental carcinogenesis, due to its broad spectrum of DNA damaging properties. In addition, bleomycin is a useful antitumor drug because of its cytotoxic properties. To investigate the influence of the antioxidant N-acetylcysteine and its metabolites glutathione and cysteine on bleomycin-induced DNA damage and more importantly to gain insight into the biological relevance of such damage, PM2 DNA was exposed to Cu(2+)-bleomycin in the presence and absence of the thiols N-acetylcysteine, glutathione and cysteine. It was found that the presence of these thiols led to a considerable enhancement of bleomycin-induced single- and double-strand breaks and a concomitant decrease in the biological activity of PM2 DNA in a dose-dependent way. A similar observation was made when ascorbic acid was used. Bleomycin showed no DNA damaging activity when PM2 DNA was pretreated with the strong Fe ion chelator desferal and its activity was strongly inhibited by the addition of Cu2+ ions or under hypoxic (N2) conditions. Cu(2+)-bleomycin under our conditions is not active by itself, but most probably after binding to DNA exchanges Cu2+ for Fe3+ bound to DNA. Fe(3+)-bleomycin is then reduced to Fe(2+)-bleomycin, a process potentiated by the added antioxidants, and subsequently activated by O2. The contribution to biological inactivation of bleomycin alone or in the presence of ascorbic acid is only approximately 15%. The contribution to lethality in the presence of thiols is higher. These results indicate that ascorbic acid only enhances the DNA damaging properties of bleomycin, whereas the thiol compounds in addition influence the type of DNA damage. The remainder of the biological inactivation is probably caused by double damage, such as single-strand breaks with closely opposed alkali-labile sites or base damage.

Gamma-radiation-induced mutation spectrum in the episomal lacI gene of Escherichia coli under oxic conditions.

In this study we have determined the mutation spectrum in the complete episomal lacI gene of Escherichia coli induced by gamma-radiation under oxic conditions. Mutants were generated by 60Co gamma-irradiation of an E. coli culture of stationary cells in LB medium, under continuous flushing with oxygen. Oligonucleotide probe analysis showed that 14% of the gamma-ray-induced mutations were located at the lacI gene hot spot at position 620-632, which is characterized by a triple repeat of the 5'-TGGC-3' sequence. Previously it was shown that about 70% of the spontaneous mutations were located at this site due to the loss or the addition of a TGGC sequence. The non-hot spot mutations were further characterized by automated sequence analysis. The results show that base pair (bp) substitutions were the main type of gamma-ray-induced mutations. Although all types of bp substitutions were observed, 74% of the bp substitutions involved C/G base pairs. C/G --> T/A and C/G --> A/T substitutions were predominant, both accounting for 35% of all bp substitutions, whereas A/T --> C/G substitutions were only seldomly observed (3%). A relatively large amount of -1 bp deletions (15% of all mutations) was detected in the gamma-ray-induced mutation spectrum, mainly affecting C/G base pairs, and 10% were deletions, ranging in size from 11 to 532 bp. It can be concluded that under oxic conditions gamma-radiation induces in E. coli mainly bp substitutions of all types but preferentially at C/G base pairs, and that the mutations tend to be randomly distributed within the lacI gene sequence.

Biological consequences of DNA damage introduced in bacteriophage PM2 DNA by hydrogen peroxide-mediated free radical reactions.

In order to study the biological consequences of DNA damage induced by H2O2-mediated free radical reactions, DNA from bacteriophage PM2 was exposed to H2O2, Fe(3+)-citrate and ascorbate either alone or in combination. Induction of DNA lesions was determined as well as the biological activity of the phage DNA. Exposure to H2O2 alone resulted in max. 0.2 single-strand breaks per molecule; in the presence of Fe(3+)-citrate, the yield was approximately 4-fold higher. Under both conditions no double-strand breaks could be detected and the biological activity was not diminished. This indicates that low levels of single-strand breaks as generated by H2O2/Fe(3+)-citrate do not inactivate PM2 DNA. Exposure to ascorbate in the presence Fe(3+)-citrate resulted in extensive induction of single-strand breaks. However, at ascorbate concentration where approximately 3 single-strand breaks per molecule were induced, again no double-strand breaks could be detected and the biological activity of the DNA was not diminished. At 5 mM ascorbate, single-strand breaks were above the detection limit. Under these conditions, 0.02 double-strand breaks were induced and the biological activity was reduced to 50%. The contribution of double-strand breaks to biological inactivation was calculated to be approximately 3%. When PM2 DNA was exposed to H2O2 in the presence of ascorbate/Fe(3+)-citrate, a typical biphasic dose-effect relationship was observed both for the induction of double-strand breaks and biological inactivation, suggesting that one or more reactive species sensitive to H2O2 play a critical role. The .OH scavenger t-butanol appeared to be relatively inefficient in protecting PM2 DNA, which may indicate that other reactive species than .OH are involved. Our data suggest that other reactive species than .OH, such as the ferryl ion, are involved in H2O2-mediated DNA damage induction and biological inactivation.

The formation of one-G deletions as a consequence of single-oxygen-induced DNA damage.

Single-stranded M13mp10 DNA containing a 144-bp mutational target sequence in the lacZ alpha gene was treated with singlet oxygen (1O2) generated by thermodissociation of the endoperoxide of 3,3'-(1,4-naphthalene-1,4-diyl)dipropionate (NDPO2). After transfection to non-SOS-induced E. coli cells, 32 mutants preselected for a mutation in the 144-bp target were collected and analyzed by DNA sequencing. One-G deletions represented the predominant type of mutation accounting for 50% of the mutations analyzed. The remaining part appeared to consist of base substitutions, i.e. G-->T transversions (34%), C-->T transitions (12.5%) and one T-->C transition (3%). Sixty percent of the mutations were found in two major mutational hotspots. We conclude that the predominant one-G deletions are due to a guanine reaction product which might be specific for 1O2.

The ambivalent role of glutathione in the protection of DNA against singlet oxygen.

Glutathione (GSH) was examined with respect to its ability to protect DNA against 1O2 damage. We have found that GSH protected, at least partly, the DNA against inactivation by 1O2. Up to 10 mM the protection increased as a function of GSH concentration. Above 10 mM the protection remained constant and less than expected on the basis of scavenging/quenching of 1O2, in contrast to the protection offered by sodium-azide. Especially at the higher concentrations of GSH the protection against the biological inactivation is accompanied by an increase in single-strand breaks and also probably lethal base damage. However, all together the data suggest that at least in the physiologically important range (0.1-10 mM) GSH is able to protect efficiently against 1O2-induced inactivating DNA damage.

C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution.

To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

Mutational specificity of oxidative DNA damage.

In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (.OH) and by exposure to singlet oxygen (1O2), released by thermo-dissociation of the endoperoxide 3,3'-(1,4-naphthalidene) dipropionate (NDPO2). We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ alpha gene. This 144 bp insert was used as a mutational target sequence. When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were gamma-irradiated in the presence of oxygen (O2; 100% .OH) or nitrous oxide (N2O; 90% .OH, 10% .H), very specific mutation spectra were found. Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type. Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one. Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed. Under N2O-1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present. To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to gamma-rays in phosphate buffer under nitrogen (55% .H, 45% .OH). Under these conditions a remarkable shift was observed from C/G-->G/C to C/G-->A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no -1 bp deletions were detected. These results strongly suggest that H radicals do not cause -1 bp deletions, but may be responsible for the observed C/G to A/T transversions. The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated. When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 bp sequence is part of M13mp10 DNA. Finally, in agreement with the observation that 1O2 reacts preferentially with guanine in DNA, a guanine is involved in most of the mutations scored after exposure of single-stranded (ss) M13mp10 DNA to NDPO2-generated 1O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Singlet oxygen-induced DNA damage: product analysis, studies of biological consequences and characterization of mutations.

The DNA lesions induced by free 1O2 and the biological and mutagenic consequences of 1O2-induced DNA damage have been studied. Using anion exchange HPLC, reverse-phase HPLC with electrochemical detection and 32P-postlabelling methods, we have shown that 1O2 reacts with 2'-deoxyguanine 3'-monophosphate (dGp) but not with any other dNp. Reaction with dGp yields a large number of products; one minor product was identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp), and a second tentatively as a formamidopyridine derivative of dGp. 8-Oxo-dGp was also found after reaction of 1O2 with single-stranded (ss) DNA, double-stranded (ds) DNA or an oligonucleotide (16-mer) having one G. With the oligonucleotide we found a second unidentified reaction product. With ss DNA, 8-oxo-dG was a much more prominent product than in the reaction of 1O2 with free dGp and the yield was about eight-fold higher than with ds DNA. This agrees with our finding that ss M13 DNA is at least 100-fold more sensitive than ds M13 DNA to biological inactivation by 1O2. The inactivation of ss M13 DNA must be largely due to 1O2-induced lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG, most of the mutations induced by 1O2 in ss or ds M13mp10 DNA occurred at a G or G/C basepair, respectively. A preference for G(C) to T(A) transversions was observed for which 8-oxo-dG might have been responsible. In ss DNA, a significant number of mutations are characterized by the fact that a G is deleted.

Contrasting effects of SH-compounds on oxidative DNA damage: repair and increase of damage.

The non-radical singlet oxygen (1O2) and the OH radical (.OH) are the major damaging oxidative species that can be generated inside cells during normal aerobic metabolism and by processes such as photosensitization. Both reactive oxygen species fulfill essential prerequisites to be a genotoxic agent. Due to their continuous production they represent an ever-present threat to all vital cellular molecules, especially DNA. As might be anticipated from the difference in character between these reactive species (non-radical versus radical) the pattern of DNA modifications caused by singlet oxygen is different from that produced by OH radicals. All cells possess an elaborate defense system against oxidative damage. This paper focuses mainly on the effect of thiols such as glutathione, which are thought to play a role as antioxidants. Under certain conditions thiols can repair chemically, probably by H-donation, some of the DNA damage caused by .OH; for instance breaks can be rather easily prevented in this way. This process will compete with fixation of damage by oxygen. However, there is ample evidence that H-atom donation does not always lead to 'correct' repair. Moreover under aerobic conditions thiyl peroxy radicals might increase DNA damage. Although the repair/fixation process could not be examined in the case of 1O2 yet, it could be demonstrated that reactive species can be formed out of the reaction of thiols with 1O2 capable of enhancing the number of DNA modifications such as 8-oxoguanine and single-strand breaks, probably arising from different pathways. Although it is quite clear that thiols are to some extent excellent antioxidants they possess unexpected properties which, depending on the conditions, can have genotoxic consequences.

Interaction of singlet oxygen with DNA and biological consequences.

To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp). A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentified reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp. Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively. A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible. In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.

Reactions of glutathione with the catechol, the ortho-quinone and the semi-quinone free radical of etoposide. Consequences for DNA inactivation.

Etoposide [4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta- D-glucopyranoside)] can be metabolized to DNA-inactivating catechol, ortho-quinone and semi-quinone free radical derivatives which may contribute to its cytotoxicity. In this paper, we examined in vitro whether glutathione (GSH), which is known to react easily with quinoid compounds, could interact with the active etoposide intermediates and in this way influence the cytotoxicity of the parent compound. To this end, reactions of GSH with the etoposide intermediates were studied, using HPLC and ESR measurements, together with the effects of GSH on the biological inactivation of single-stranded (ss) and double-stranded (RF) phi X174 DNA by these compounds. From the results it could be determined that: (a) GSH does not react with the catechol and, as a consequence, has no effect on the reaction of this intermediate of etoposide with ss and RF phi X174 DNA; (b) GSH reacts with the ortho-quinone most likely by formation of a conjugate and by two-electron reduction to the catechol, resulting in a partial protection of ss and RF phi X174 DNA against inactivation by this species; and (c) GSH protects ss phi X174 DNA against inactivation by the semi-quinone free radical of etoposide probably by conjugation with this species.

Inactivation and repair of double-stranded DNA damaged by the aziridinyl nitroimidazole RSU 1069.

Incubation of RSU 1069 in the presence of biologically active double-stranded phi X174 DNA resulted in, depending on pH, ionic strength and concentration of drug, inactivation of the DNA. A variety of lesions are induced including a high number of single-strand breaks and alkali-labile lesions, which are at most partly lethal. The main inactivating damage consists probably of base damage, induced by alkylation. A considerable part of the damage induced by RSU 1069 can be repaired by the various repair enzymes of the bacterial host of the phi X174 DNA. Finally the damage (pattern) depends considerably on the ionic composition of the reaction solution, which can be explained by an equilibrium model presented in this paper.

Modulation by D,L-buthionine-S,R-sulphoximine of etoposide cytotoxicity on human non-small cell lung, ovarian and breast carcinoma cell lines.

Treatment with 25 mumol/l D,L-buthionine-S,R-sulphoximine (BSO) for at least 24 h depleted glutathione (GSH) in human non-small cell lung (SW-1573), ovarian (A2780) and breast carcinoma (MCF-7) cell lines to about 20% of control, and was accompanied by a 2-fold potentiation of the cytotoxicity of etoposide, doxorubicin and cisplatin. Cellular etoposide, but not doxorubicin or cisplatin, concentrations were increased 2-fold due to decreased efflux. This occurred independently of the presence of BSO during 1 h of etoposide exposure, but required prolonged exposure to BSO (at least 24 h). Energy depletion as well as cotreatment, but not pretreatment, of the cells with daunomycin, doxorubicin, vinblastine or vincristine increased cellular etoposide accumulation. Treatment of control cells with verapamil caused similar changes in etoposide cytotoxicity and cellular pharmacokinetics as GSH depletion, but did not further increase etoposide cytotoxicity and accumulation in GSH-depleted cells. Etoposide efflux may have been inhibited, not because of (competitive) inhibition by BSO or disturbance of the energy required for this process, but probably because of plasma membrane alterations.

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

Mutational specificity of gamma-rays differs for the same target in plasmid DNA and double-stranded (RF) M13 bacteriophage DNA.

In the lacZ alpha gene of a pUC plasmid a 144 bp insert was cloned as target for mutagenesis. Irradiation of the plasmid in a diluted aqueous solution by 60Co gamma-rays under oxic conditions leads to a very specific mutation spectrum. The predominant type of mutation was a C/G to A/T transversion (29 out of 47 mutants) whereas C/G to G/C transversions were found 7 times and C/G to T/A transitions 10 times. Only one frameshift could be observed which was a deletion of an A/T base pair. The mutations were not randomly distributed along the mutation target but show a strong preference for a certain DNA sequence in which two thirds of the mutations were scored. In this DNA area a hotspot (24 of the 47 mutants) for mutagenesis was located and within 6 bp next to this hotspot another seven mutations were scored. The mutation spectrum in the same mutation target as part of double-stranded (RF) M13 phage was published before. In both systems the mutational hotspot is located at the same site, but the predominant type of mutation is different. In the M13 system the C/G to G/C transversion was the most important event.

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.