Biomedical subjects

M V Lafleur

Publications and source records attributed to M V Lafleur.

At least 19 recordsLinked to original sources

PubMed2001

The importance of using absolute mutant frequencies to compare mutation spectra.

Because damage to the cellular DNA is very hazardous for a cell, it is important to identify compounds, which can cause DNA damage. To investigate the mutagenic effect of a particular agent of interest, usually mutation spectra are determined in a selected target gene. The most commonly used method to compare different mutation spectra with each other, is the comparison of the percentages of each type of mutation. In this paper, it is emphasized that comparison of percentages can lead to incorrect conclusions and therefore another determinant, the absolute mutant frequency, should be used.

DNA↗

PubMed2000

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10.

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).

Bacteriophage M13↗

PubMed2000

Mutagenic effect by phenylalanine during gamma-irradiation of plasmid DNA in aqueous solution under oxic conditions.

Irradiation of DNA in aqueous solution or in cells with gamma-rays results in different mutational spectra, indicating that in both situations different patterns of DNA damages are induced. One of the causes for these different types of damages might be the formation of secondary, organic radicals, if cells are irradiated. Some organic compounds, including the amino acid phenylalanine, are well known to produce radicals during irradiation. Under oxic conditions these secondary radicals react with oxygen, thus forming peroxyl radicals which can be very harmful to DNA, and which may, therefore, induce DNA damage leading to mutations. This study examines the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions. The results indicate that the formation of phenylalanine radicals influences the types of induced mutations in the gamma-radiation-induced mutation spectrum. The most prominent difference is the increase in G:C to T:A transversions and the decrease in G:C to A:T transitions in the presence of phenylalanine. Further, it appears that the gamma-radiation-induced mutation spectrum after irradiation of DNA in aqueous solution is more comparable to the intracellular gamma-radiation-induced mutation spectrum in E. coli cells, if phenylalanine is present during irradiation. Therefore, these results suggest that the presence of phenylalanine during irradiation of DNA in aqueous solution gives a better impression of gamma-radiation-induced mutations in bacterial systems than water only.

DNA↗

PubMed2000

The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene.

Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E. coli strain (JM105). The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient. Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the formation of mutations.

Adenosine Triphosphatases↗

PubMed1999

The influence of formamidopyrimidine-DNA glycosylase on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZ alpha gene.

Base excision repair (BER) is a very important repair mechanism to cope with oxidative DNA damage. One of the most predominating oxidative DNA damages after exposure to ionizing radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This damage is repaired by formamidopyrimidine-DNA glycosylase (Fpg), a DNA glycosylase which is part of BER. Correct repair of 8oxoG is of great importance for cells, because 8oxoG has strong miscoding properties. Mispairing of 8oxoG with adenine instead of cytosine results in G:C to T:A transversion mutations. To determine the effect of a Fpg-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene, double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target, was irradiated with gamma-rays in aqueous solution under oxic conditions. Subsequently, the DNA was transfected into a wild-type Escherichia coli strain (JM105) and an isogenic Fpg-deficient E. coli strain (BH410). Although the overall spontaneous mutation spectra between JM105 and BH410 seemed similar, remarkable differences could be observed when the individual base pair substitutions were viewed. The amount of C to A transversions, which are most probably caused by unrepaired 8oxoG, has increased 3. 5-fold in the spontaneous BH410 spectrum. When the gamma-radiation-induced mutation spectra of JM105 and BH410 were compared, there was even a larger increase of C to A transversions in the BH410 strain (7-fold). We can therefore conclude that the straightforward approach used in this study confirms the importance of Fpg in repair of gamma-radiation-induced damage, and most probably especially in the repair of 8oxoG.

DNA Mutational Analysis↗

PubMed1999

The presence of traces of iron and copper ions during gamma-irradiation does not result in clear mutational hot spots in the lacI gene.

Oxidative radicals, which are produced during ionizing irradiation of DNA in water, damage the DNA and may result in mutations, which are in general randomly distributed. Alternatively, the addition of transition metal ions, like iron or copper, to DNA in combination with H(2)O(2) and a reducing agent also results in the production of oxidative radicals. Due to binding of the transition metal ions to DNA, the production of these radicals is very local, and results in a mutational spectrum in which the mutations are not randomly distributed. If transition metal ions are complexed to the DNA during irradiation, and react with radiation-induced species such as hydrogen peroxide, site-specific formation of.OH radicals on these sites may occur, leading to the formation of mutational hot spots. This study examines the influence of the presence of traces of iron or copper ions during gamma-irradiation of plasmid DNA in water, on the possible formation of mutational hot spots in the lacI gene. Comparison of the mutational spectra, after irradiation in the presence or in the absence of transition metal ions, shows that there are indeed relatively more positions in the lacI gene where more than one mutation occurs, suggesting formation of mutational hot spots in the presence of transition metal ions. However, the appearance of these hot spots is rather weak. Although in all three mutational spectra G:C to A:T mutations are predominant, there are also some differences between the types of mutations in these spectra. These differences in mutational spectra might reflect the different preferences of iron and copper ions to bind specific sites in the DNA. Indeed, there appears to be a high association of mutations at CC or GG sites in the mutational spectrum in the presence of copper ions, confirming the observation that copper binds preferably at two adjacent guanines in the DNA. It can be concluded from this study that the presence of small amounts of transition metal ions during gamma-irradiation influences the types and distribution of gamma-radiation-induced mutations, although no major mutational hot spots can be observed.

Bacterial Proteins↗

PubMed1998

Influence of the UV-activated SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene.

Previous studies of our group have shown that intracellular or extracellular gamma-irradiation of the lacI gene results in different mutational spectra. One cause for these differences might be the error-prone SOS response, which is activated in the intracellular situation by gamma-irradiation but not in the extracellular situation. Since UV-radiation is a well-established strong inducer of the SOS response, we used bacterial host cells, pretreated with UV-light to study the influence of the SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene in the extracellular situation. If the SOS response was activated, mutations on A:T base pairs and frameshift mutations accounted for 16% and 12% of all mutations, respectively, but they were hardly detected in the absence of an induced SOS response. G:C to T:A transversions increased from 14% to 24% in the presence of an activated SOS response. We can therefore conclude from this study, that SOS-induction of host cells by UV-light influences the extracellular mutation spectrum in the lacI gene, with respect to mutations on A:T base pairs, G:C to T:A transversions and frameshift mutations. This conclusion is supported by the fact that the previously obtained intracellular gamma-radiation-induced mutation spectrum in the lacI gene, in which the SOS response is also involved, shows great similarities with the extracellular mutation spectrum in the presence of an activated SOS response in this study.

Bacterial Proteins↗

PubMed1998

Characterization of DNA damage induced by gamma-radiation-derived water radicals, using DNA repair enzymes.

PURPOSE: To characterize the DNA damage profiles due to gamma-radiation induced water radicals. MATERIALS AND METHODS: Double stranded (ds) phiX174 DNA was irradiated in aqueous solution with gamma-rays under different gassing conditions (O2, N2O or N2) and the damage profiles were determined using DNA repair enzymes. RESULTS: The DNA damage profile under O2 is characterized by about equal numbers of direct single-strand breaks (ssb) and Fpg sensitive sites, whereas endonuclease III and exonuclease III sites are formed in lower amounts. The DNA damage profiles under N2O and N2 in phosphate buffer consist predominantly of direct single-strand breaks. Fpg sensitive sites dominate the DNA damage profile under N2 in phosphate buffer in the presence of the radical scavenger 2-methyl propan-2-ol, where (almost) only .H atoms are present. CONCLUSIONS: Both .OH radicals and .H atoms induce direct single-strand breaks, but .OH radicals are the most effective ones. Fpg sensitive sites are induced in high amounts by both .OH radicals and H atoms, but when both types of radicals are present, the formation of Fpg sensitive sites is prevented. Hydrated electrons (e(aq)-) contribute to inactivation of DNA, although only a very small fraction of the e(aq)- is involved in this process.

Bacteriophage phi X 174↗

PubMed1996

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.

Bacteriophage M13↗

PubMed1996

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.

Aerobiosis↗

PubMed1996

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.

Bacteriophage M13↗

PubMed1996

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.

Ascorbic Acid↗

PubMed1996

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.

Acetylcysteine↗

PubMed1994

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.

Bacteriophage M13↗

PubMed1994

Role of oxidative DNA damage in the mechanism of fecapentaene-12 genotoxicity.

Fecapentaene-12 (FP-12), a fecal unsaturated, ether-linked lipid excreted by most human individuals in Western populations, has been found to be a potent genotoxin in mammalian cells. Its mechanism of genotoxicity may be mediated by oxygen radical-induced DNA damage or by direct DNA alkylation, of which the relative importance remains to be determined. In the present study, induction of oxidative genetic damage by FP-12 has been investigated, in combination with the biological inactivation of single-stranded bacteriophage phi X-174 DNA. It was shown that formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), a marker for oxidative DNA damage, is induced dose dependently by FP-12 in 2'-deoxyguanosine (dG). It was demonstrated by application of radical scavengers that production of both the superoxide anion and singlet oxygen may be involved in the induction of 8-oxodG. The effect of OH radical scavenging appeared to be less pronounced. Enzymatic peroxidation of FP-12, which has been demonstrated to stimulate oxygen radical formation, was found to increase the hydroxylation ratio in dG, an effect which was less pronounced in single-stranded DNA and even absent in double-stranded DNA. No induction of 8-oxodG was observed after exposure of human skin fibroblasts to 60 microM FP-12 for 3 h in vitro. It was concluded that the induction of 8-oxodG by FP-12 is determined by the accessibility of the guanine molecule rather than the rate of oxygen radical formation. Although free radical formation is known to be stimulated by enzymatic peroxidation of FP-12, the inactivation of phi X-174 DNA spontaneously induced by FP-12 was found to be reduced by application of peroxidases. This furthermore demonstrates that the increased formation of reactive oxygen species by enzymatic peroxidation of FP-12 does not directly relate to increased induction of genotoxic effects. The fact that addition of radical scavengers shows limited effects on the inactivation of phi X-174 DNA suggests that the contribution of oxidative DNA damage to the genotoxic potential of FP-12 is only of minor importance.

Animals↗

PubMed1994

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.

Azides↗

PubMed1993

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.

Bacteriophage M13↗

PubMed1993

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)

Base Sequence↗