Mutational signatures in tumours induced by high and low energy radiation in Trp53 deficient mice.

Ionising radiation (IR) is a recognised carcinogen responsible for cancer development in patients previously treated using radiotherapy, and in individuals exposed as a result of accidents at nuclear energy plants. However, the mutational signatures induced by distinct types and doses of radiation are unknown. Here, we analyse the genetic architecture of mammary tumours, lymphomas and sarcomas induced by high (56Fe-ions) or low (gamma) energy radiation in mice carrying Trp53 loss of function alleles. In mammary tumours, high-energy radiation is associated with induction of focal structural variants, leading to genomic instability and Met amplification. Gamma-radiation is linked to large-scale structural variants and a point mutation signature associated with oxidative stress. The genomic architecture of carcinomas, sarcomas and lymphomas arising in the same animals are significantly different. Our study illustrates the complex interactions between radiation quality, germline Trp53 deficiency and tissue/cell of origin in shaping the genomic landscape of IR-induced tumours.

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80-100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea.

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or acetate as carbon and energy sources. This method provides an avenue for the first step in identifying new gene functions: associating a genetic mutation with a reproducible phenotype. Mutations in bona fide methanogenesis genes such as corrinoid methyltransferases and proton-translocating F420H2:methanophenazine oxidoreductase (Fpo) were also generated, opening the door to in vivo functional complementation experiments. Irradiation-based mutagenesis such as from ultraviolet (UV) light, combined with modern genome sequencing, is a useful procedure to discern systems-level gene function in prokaryote taxa that can be axenically cultured but which may be resistant to chemical mutagens.

Murine melanomas accelerated by a single UVR exposure carry photoproduct footprints but lack UV signature C>T mutations in critical genes.

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. C>T and CC>TT changes make up the overwhelming majority of single base substitutions (SBS) in MM DNA, as both precursor melanocytes and melanocytic lesions have incurred incidental exposures to sunlight. To study the mutagenic mechanisms by which acute sunburn accelerates MM, we sequenced the exomes of spontaneous and neonatal UVB-induced Cdk4-R24C::Tyr-NRASQ61K mouse MMs. UVR-induced MMs carried more SBSs than spontaneous MMs, but the levels of genomic instability, reflected by translocations and copy number changes, were not different. C>T/G>A was the most common SBS in spontaneous and UVR-induced MMs, only modestly increased in the latter. However, they tended to occur at the motif A/GpCpG (reflecting C>T transition due to spontaneous deamination of cytosine at CpG) in spontaneous MMs, and T/CpCpC/T (reflecting the effects of pyrimidine dimers on either side of the mutated C) in UVR-induced MMs. Unlike MMs associated with repetitive exposures, we observed no CC>TT changes. In addition, we also found UVR 'footprints' at T>A/A>Ts (at NpTpT) and T>C/A>G (at CpTpC). These footprints are also present in MMs from a chronic UVR mouse model, and in some human MMs, suggesting that they may be minor UVR signature changes. We found few significantly somatically mutated genes (~6 per spontaneous and 15 per UVR-induced melanoma) in addition to the Cdk4 and NRAS mutations already present. Trp53 was the most convincing recurrently mutated gene; however, in the UVR-induced MMs no Trp53 mutations were at C>T/G>A, suggesting that it was probably mutated during tumour progression, not directly induced by UVR photoproducts. The very low load of recurrent mutations convincingly induced by classical UVB-induced dimer photoproducts may support a role for cell extrinsic mechanisms, such as photoimmunosuppression and inflammation in driving MM after acute UVB exposure.

Communication: UV photoionization of cytosine catalyzed by Ag(+).

The photo-induced damages of DNA in interaction with metal cations, which are found in various environments, still remain to be characterized. In this paper, we show how the complexation of a DNA base (cytosine (Cyt)) with a metal cation (Ag(+)) changes its electronic properties. By means of UV photofragment spectroscopy of cold ions, it was found that the photoexcitation of the CytAg(+) complex at low energy (315-282) nm efficiently leads to ionized cytosine (Cyt(+)) as the single product. This occurs through a charge transfer state in which an electron from the p orbital of Cyt is promoted to Ag(+), as confirmed by ab initio calculations at the TD-DFT/B3LYP and RI-ADC(2) theory level using the SV(P) basis set. The low ionization energy of Cyt in the presence of Ag(+) could have important implications as point mutation of DNA upon sunlight exposition.

Frequency and features of TP53 mutation in 30 Chinese patients with sporadic basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is a prevalent form of nonmelanoma skin cancer. Although numerous studies in white populations suggest that mutations in the TP53 gene play an important role in the development of BCC, it is not clear whether this is also the case in East Asian populations such as in China. AIM: To investigate the frequency and the features of TP53 mutation in sporadic BCC in a Chinese population. METHODS: In total, 30 patients with sporadic BCC, who had previously taken part in a study on PTCH1 mutations, were enrolled. BCC and control cells were obtained by laser-capture microdissection, and DNA was amplified and sequenced for analysis of TP53 mutations. RESULTS: In the 30 BCC samples, 6 TP53 point mutations were found (frequency of 20%), and 4 of these 6 mutations had ultraviolet (UV)-specific alterations. Combining these results with those of the previous study on PTCH1 mutations, we found that two patients with had three types of genetic alterations (each had two PTCH1 mutations and one TP53 point mutation). A further two patients each had one PTCH1 mutation and one UV signature TP53 mutation. In addition, the total number of UV-specific mutations of PTCH1 and TP53 accounted for 20% of the total patient group. CONCLUSIONS: The incidence of TP53 mutation in BCC in our Chinese subjects was lower than that reported for white populations. Many of the patients carried mutations of other genes in addition to of TP53. The majority of TP53 mutations were UV-induced specific alterations. However, the results of the two studies on TP53 and PTCH1 indicated that the incidence of UV-specific mutations is much lower in Chinese than in white populations.

[Radiation biology of structurally different Drosophila genes. Report V. The cinnabar gene: general and molecular characteristics of its radiomutability].

The results of the genetic, cytogenetic and molecular analysis of the recessive mutations at the small, lying close to the centromere, cinnabar (cn) gene of Drosophila melanogaster induced by γ-rays of 60Co (doses 5-60 Gy) or 0.85 MeV fission neutrons (doses 2.5-20 Gy) in the mature sperm of the wild-type males from the laboratory line D32 are presented. The whole spectrum of the cn mutations induced by different quality radiation is found to be the same and consists of the two main-distinct classes such as gene/point and gene/chromosome mutations either of which includes the array of the subclasses (gene/point simple or complex mutations and chromosome rearrangements detected as F1 cn mutants with dominant sterility of multilocus deletions involving the cn gene wholly). The induction rate of both mutation classes is found to be increased linearly with dose of low- and high-LET radiation and the RGE values of neutrons are 1.0 and 4.0 for the gene/point and gene/chromosome mutations respectively. According to the data of the molecular analysis, 28 out of 59 (47.5%) γ-ray- and neutron-induced gene/point cn mutations studied are found to have the intragenic DNA alterations detected by PCR technique as a loss of the single or two adjacent fragments-amplicons non-randomly located at the 5'- or 3'- end of the gene map. Essentially, 10 out of 48 (20.8%) γ-ray-and 3 out of 11 (27.3%) neutron-induced gene/point mutations are found to show the same molecular "phenotype" (the loss of the two adjacent fragments at the 3'- end of the gene map) as that in the cn1 allele-marker from the maternal tester-line KL with the females of which the irradiated males were crossed. Among the putative recombination-based genetic processes underlying the exchange between the cn1 and damaged cn(+32) alleles, the gene conversion in the "gonomeric" nucleus of the zygote seems to be the most likely such processing. The established features of the cn gene radiomutability are compared with those earlier described for the other small gene black+ located in the middle of the 2L chromosome.

[Radiation biology of structurally different drosophila genes. Report IV. The black gene: sequencing of the "point" mutations and recombination mechanisms of their processing].

As it has been ascertained in our large-scale experiments with Drosophila specific five-loci test [1], the radio- mutability of the black gene is unusual at lest in two respects: 1) fission neutrons are strangely more efficient than γ-rays in the gene/point mutation induction and 2) a lot of gene/point black mutations have the DNA alterations not detected by PCR (so-called PCR(+)-mutants). To verify the hypothesis that neutrons induce more efficiently than γ-rays the small structural DNA changes which fail to notice the PCR, sequence ana- lysis of 8 neutron-, 8 γ-ray-induced and 3 spontaneous (from instable D32 line) black gene/point PCR(+)-mutations was performed. As controls, sequences of the test-allele black1, as well as irradiated black(+32) and black(+18) alleles were analyzed. In black1 the replacement of four bases (ATCC) by an insertion (TACCTACC) at position +530 (exon 1) results in a frameshift. There were also 27 single base pair substitutions compared to the control black(+32) or black(+18) sequence. Further, 6 γ-ray- and one neutron-induced black mutants displayed the small deletions/insertions and transversion (G --> T) which led to the stop-codon in one case. These nucleotide changes thought to be the result of γ-ray-induced processing by the NHEJ, SSA or MMR repair pathways which act in the early zygote ahead of the first (gonomeric) nuclear division. Remarkably, 3 spontaneous, 2 γ-ray- and 7 neutron-induced black mutants were found to have the sequence alterations intrinsic to the black allele showing that interallelic recombination (gene conversion) seems to be a major pathway of processing of the gross DNA lesions by acting of the HR, SDSA or BIR repair systems in zygote after the gonomeric division. Substantially, the frequency of conversion events for the neutron-induced DNA lesions was found to be 3.5 time as high as for γ-ray-induced ones. The genetic impact of the radiation-induced conversion events in zygotic nucleus leading to the mutant allele homozygosity (reconstitution of homozygosity, ROH) is discussed.

The point mutation induced by the low-energy N+ ion implantation in impatiens balsamine genome.

To investigate the effect of genome mutations induced by low energy ions implantation in higher plants, genome mutation of Impatiens balsamine mutant induced by low energy N+ ion implantation were analyzed by the RAPD, ISSR and genome sequence. Six out of the 121 ISSR primers and 6 out of the 135 RAPD primers showed that polymorphism ratios between mutants and wild type were 4.96% and 2.89%, respectively. Sequence analysis revealed that base deletions, insertions, and substitutions were observed in the mutant genome comparable to wild type. N+ induced point mutations were mostly base substitution (77.4%), no duplication, long fragments insertions and deletions was found. In all point mutation, adenine (A) was most sensitive to the N+ ion implantation in impatiens. The transition was mainly A --> guanine (G) (15.90%) and thymine (T) --> cytosine (C) (12.55%). Transversion happened in A <--> T (16.74%), which much higher than C <--> G(5.02%), G <--> T(6.69%), A <--> C (7.11%) bases. These findings indicate that low energy ions being a useful mutagen were mostly cause the point mutation in impatiens.

[Radiation biology of structurally different Drosophila genes. Report III. The black gene: general and molecular characteristics of its radiomutability].

The results of the genetic, cytogenetic and molecular analysis of the nature of heritable recessive mutations at the small black (b) gene of Drosophila melanogaster induced by different doses (5-10 Gy) of 60Co gamma-rays and 0.85 MeV fission neutrons in the mature sperms of the wild-type males from the laboratory line D32 are presented. The whole spectrum of the b mutations induced by radiation of different quality is found to be the same and consists of the two main classes such as gene/point and gene/chromosome mutations, the latter of which include the whole-genomic, infra- or inter-chromosomal rearrangements involving the b gene. The induction rate of both mutation classes is found to be increased linearly with a dose of low- and high-LET radiation and the effectiveness of neutrons is 2.7 and 4.6 as large as that of gamma-rays under the gene/point and gene/chromosome mutation induction, respectively. Essentially, the molecular alterations underlying 65 gamma-ray- and neutron-induced gene/point b mutations are found not to be detected by the PCR technique. These and other established features of the b gene radiomutability are drastically different from those of another larger vestigial gene described earlier. The nature of these differences is discussed within the framework of the current notion of different biological organization of the two genes mentioned above and of the track structure theory as well.

Escherichia coli mutants induced by multi-ion irradiation.

Wild-type Escherichia coli K12 strain W3110 was irradiated by 10 keV nitrogen ions. Specifically, irradiation was performed six times by N(+) ions, followed by the selection of lac constitutive mutants, and each time a stable S55 mutant was produced. By sequencing the whole genome, the fine map of S55 was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels), and nine structural variations (SVs) were found in the S55 genome. Among the 18 SNPs, 11 are transversional from A, T or C to G, accounting for 55.6% of point mutations. GCCA insertion occurs in the target gene lacI. Four SNPs, including three in rlpB and one in ygbN, are connected with cell envelope and transport. All nine structural variations of S55 are deletions and contain insertion sequence (IS) elements. Six deleted SVs contain disrupted ISs, nonfunctional pseudogenes, and one more 23 252 bp SV in the Rac prophage region. Overall, our results show that deletion bias observed in E. coli K12 genome evolution is generally related to the deletion of some nonfunctional regions. Furthermore, since ISs are unstable factors in a genome, the multi-ion irradiations that caused these deleted fragments in S55 turn out to be beneficial to genome stability, generating a wider mutational spectrum. Thus, it is possible that the mutation of these genes increases the ability of the E. coli genome to resist etch and damage caused by ion irradiation.

Molecular analysis of point mutations in a barley genome exposed to MNU and gamma rays.

We present studies aimed at determining the types and frequencies of mutations induced in the barley genome after treatment with chemical (N-methyl-N-nitrosourea, MNU) and physical (gamma rays) mutagens. We created M(2) populations of a doubled haploid line and used them for the analysis of mutations in targeted DNA sequences and over an entire barley genome using TILLING (Targeting Induced Local Lesions in Genomes) and AFLP (Amplified Fragment Length Polymorphism) technique, respectively. Based on the TILLING analysis of the total DNA sequence of 4,537,117bp in the MNU population, the average mutation density was estimated as 1/504kb. Only one nucleotide change was found after an analysis of 3,207,444bp derived from the highest dose of gamma rays applied. MNU was clearly a more efficient mutagen than gamma rays in inducing point mutations in barley. The majority (63.6%) of the MNU-induced nucleotide changes were transitions, with a similar number of G>A and C>T substitutions. The similar share of G>A and C>T transitions indicates a lack of bias in the repair of O(6)-methylguanine lesions between DNA strands. There was, however, a strong specificity of the nucleotide surrounding the O(6)-meG at the -1 position. Purines formed 81% of nucleotides observed at the -1 site. Scanning the barley genome with AFLP markers revealed ca. a three times higher level of AFLP polymorphism in MNU-treated as compared to the gamma-irradiated population. In order to check whether AFLP markers can really scan the whole barley genome for mutagen-induced polymorphism, 114 different AFLP products, were cloned and sequenced. 94% of bands were heterogenic, with some bands containing up to 8 different amplicons. The polymorphic AFLP products were characterised in terms of their similarity to the records deposited in a GenBank database. The types of sequences present in the polymorphic bands reflected the organisation of the barley genome.

Mutagenic mechanisms in leukemia and cancer: a new concept Cytosine lack could be as mutagenic as cytosine deamination.

The molecular mechanisms involved in mutagenesis caused by nitrous acid, ultraviolet light and cytosine methylation are reviewed. All three mutagens lead to a G=C --> A=T point mutation in the DNA molecule and the biochemical basis of these mutations in each case is the deamination of cytosine to uracil. It is shown that the lack of cytosine could be as mutagenic as the deamination of cytosine and it is shown schematically how cytosine lack could give rise to a population of genetically abnormal cells which are completely incapable of any degree of differentiation; a state perhaps reminiscent of the most acute of leukemias and the most anaplastic of cancers. A metabolic block in the amination of uracil to cytosine is suggested as a possible cause of the cytosine lack, a block which is unique in that it incorporates a mutagenic mechanism.

Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS.

Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction.

Biological consequences of 8-methoxypsoralen-photoinduced lesions: sequence-specificity of mutations and preponderance of T to C and T to a mutations.

Psoriatic patients undergoing psoralen plus ultraviolet radiation (PUVA) therapy are susceptible for squamous cell carcinoma and melanoma of the skin. To investigate the etiological relevance of PUVA for these diseases, we performed mutation spectrometry on the cII transgene in mouse embryonic fibroblasts treated with a single or split PUVA dose (PUVA-I or PUVA-II, respectively). Both treatments were significantly mutagenic as they increased the cII mutant frequency up to 3.7-fold over background, and produced different mutational spectra from that derived spontaneously (p<0.01), but not from one another. The signature of induced mutations, i.e., T to C transitions and T to A transversions with significant site-specificities, i.e., adjacent to T bases at the 3'-neighboring side and to pyrimidines at the 5'-neighboring side, was more pronounced after PUVA-II treatment. Also, the overall mutations occurring at T bases with the same site-specificities were more prevalent after PUVA-II treatment. The characteristic PUVA-induced mutations predominate in the p53 mutational spectrum in controlled in vivo test systems or in high-dose PUVA-treated patients, and also are easily recognizable in the overall PUVA-treated patients. We conclude that PUVA-induced mutagenesis is initiated by PUVA-I treatment and subsequently, augmented by PUVA-II treatment, leaving a unique mutational signature on the cII transgene. The signature mutations of PUVA are discernible in the p53 mutational spectrum in PUVA-treated patients but complex exposure to other therapeutic/environmental carcinogens also leads to the frequent occurrence of other types of mutations in this population.

Effects of high static magnetic field exposure on different DNAs.

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.

Ultraviolet exposure as the main initiator of p53 mutations in basal cell carcinomas from psoralen and ultraviolet A-treated patients with psoriasis.

Basal cell carcinoma, the most frequent skin cancer in humans, is often linked to chronic sun exposure. In psoralen and ultraviolet A-treated psoriatic patients, basal cell carcinomas may occur even more frequently; however, the exact etiology and mechanisms of tumorigenesis in psoriatic patients are unclear because psoralen and ultraviolet A is not only a carcinogen but also an immunosuppressor and because psoralen and ultraviolet A-treated psoriatic patients often have other (co)carcinogenic risk factors (e.g, therapeutic exposure to ultraviolet B, X-ray radiation, arsenic, tar, and/or chemotherapeutic agents such as methotrexate). In this study, we analyzed the DNA of 13 basal cell carcinomas from five psoralen and ultraviolet A-treated psoriatic patients for mutations of the p53 tumor suppressor gene. DNA sequencing revealed a total of 11 mis-sense, two non-sense, and four silent mutations in seven of the 13 basal cell carcinomas (54%). Of the 13 total mis-sense or non-sense mutations, 12 (92%) occurred at dipyrimidine sites and nine (69%) were of the ultraviolet fingerprint type (eight C-->T transitions and one CC-->TT transition). Three of the C-->T transitions occurred at dipyrimidine sites opposite a 5'-TpG sequence (a potential psoralen-binding site and target for psoralen and ultraviolet A mutagenesis). Thus, whether these mutations were induced by ultraviolet or psoralen and ultraviolet A was not clear. In addition, two other mutations (15%) occurred at 5'-TpG sites, one (8%) occurred at a 5'-TpA site (the most frequent site of psoralen binding and mutagenesis in cell and murine studies), and one (8%) involved a G-->T transversion. These results suggest that (i) the major initiator of p53 mutations in basal cell carcinoma in psoralen and ultraviolet A-treated psoriasis patients is environmental and/or therapeutic ultraviolet(B) exposure, and that (ii) psoralen and ultraviolet A itself causes only a smaller portion of p53 mutations in psoralen and ultraviolet A-associated basal cell carcinomas.

Possible cause of G-C-->C-G transversion mutation by guanine oxidation product, imidazolone.

BACKGROUND: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G-C-->T-A and G-C-->C-G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G-C-->T-A transversion mutations. Although the number of G-C-->C-G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G-C-->C-G transversions is not known. RESULTS: To determine which oxidative products are responsible for G-C-->C-G transversion mutations, we photooxidized 5'-d(AAAAAAGGAAAAAA)/5'-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4 degrees C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2'-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4H-imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz-G base pairs are formed. The 'reverse' approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz-G base pair. CONCLUSIONS: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz-G base pair formation can explain the G-C-->C-G transversion mutations that appear under oxidative conditions.