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ABSTRACT: UVA1 exerts its genotoxicity on mammalian skin by producing cyclobutane pyrimidine dimers (CPDs) in DNA and preferentially inducing solar-UV-signature mutations, C → T base substitution mutations at methylated CpG-associated dipyrimidine (Py-mCpG) sites, as demonstrated previously using a 364 nm laser as a UVA1 source and lacZ-transgenic mice that utilize the transgene as a mutational reporter. In the present study, we confirmed that a broadband UVA1 source induced the same mutation profiles in mouse epidermis as the UVA1 laser, generalizing the previous result from a single 364 nm to a wider wavelength range of UVA1 (340-400 nm). Combined with our previous data on the mutation spectra induced in mouse epidermis by UVB, UVA2 and solar UVR, we proved that the solar-UV-signature mutation is commonly observed in the wavelength range from UVB to UVA, and found that UVA1 induces this mutation more preferentially than the other shorter wavelength ranges. This finding indicates that the solar-UV-signature mutation-causing CPDs, which are known to prefer Py-mCpG sites, could be produced with the energy provided by the longer wavelength region of UVR, suggesting a photochemical reaction through the excitation of pyrimidine bases to energy states that can be accomplished by absorption of even low-energy UVR. On the other hand, the lower proportions of solar-UV-signature mutations observed in the mutation spectra for UVB and solar UVR indicate that the direct photochemical reaction through excited singlet state of pyrimidine bases, which can be accomplished only by high-energy UVR, is also involved in the mutation induction at those shorter wavelengths of UVR. We also found that the solar-UV signature prefers 5'-TCG-3' to 5'-CCG-3' as mutational target sites, consistent with the fact that UVA induces CPDs selectively at thymine-containing dipyrimidine sites and that solar UVR induces them preferably at Py-mCpG sites. However, the mutation spectrum in human p53 gene from non-melanoma skin cancers shows the opposite preference for 5'-CCG-3' sites. This apparent discrepancy in the site preference seems to result from the lack of 5'-TCG-3' sites mutable to missense mutations on the nontranscribed strand of human p53 gene, which should be evolutionally acquired under selective pressure from the sun.
Photochemical and Photobiological Sciences 03/2013; · 2.58 Impact Factor
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Hironobu Ikehata,
Shoichi Higashi,
Shingo Nakamura,
Yasukazu Daigaku,
Yoshiya Furusawa,
Yasuhiro Kamei,
Masakatsu Watanabe,
Kazuo Yamamoto,
Kotaro Hieda,
Nobuo Munakata,
Tetsuya Ono
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ABSTRACT: UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.Journal of Investigative Dermatology advance online publication, 14 February 2013; doi:10.1038/jid.2012.504.
Journal of Investigative Dermatology 02/2013; · 6.31 Impact Factor
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ABSTRACT: In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 03/2012; 734(1-2):50-5. · 2.85 Impact Factor
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ABSTRACT: During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression.
Experimental Cell Research 03/2012; 318(5):623-31. · 3.58 Impact Factor
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Kenichi Masumura,
Yasuteru Sakamoto,
Megumi Ikeda,
Yasuo Asami,
Tetsuya Tsukamoto, Hironobu Ikehata,
Yuichi Kuroiwa,
Takashi Umemura,
Akiyoshi Nishikawa,
Masae Tatematsu,
Tetsuya Ono,
Takehiko Nohmi
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ABSTRACT: Tumor development in the skin may be a multistep process where multiple genetic alterations occur successively. The p53 gene is involved in genome stability and thus is referred to as "the guardian of the genome." To better understand the antigenotoxic effects of p53 in ultraviolet light B (UVB)-induced mutagenesis, mutations were measured in the epidermis of UVB-irradiated p53(+/+) and p53(-/-) gpt delta mice. In the mouse model, point mutations and deletions are separately identified by the gpt and Spi(-) assays, respectively. The mice were exposed to UVB at single doses of 0.5, 1.0, or 2.0 kJ/m(2) . The mutant frequencies (MFs) were determined 4 weeks after the irradiation. All doses of UVB irradiation enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no significant differences in gpt MFs and the mutation spectra between p53(+/+) and p53(-/-) mice. The predominant mutations induced by UVB irradiation were G:C to A:T transitions at dipyrimidines. In contrast, in unirradiated p53(-/-) mice, the frequencies of Spi(-) large deletions of more than 1 kb and complex-type deletions with rearrangements were significantly higher than those of the Spi(-) large deletions in p53(+/+) counterparts. The specific Spi(-) mutation frequency of more than 1 kb deletions and complex types increased in a dose-dependent manner in the p53(+/+) mice. However, no increase of such large deletions was observed in irradiated p53(-/-) mice. These results suggest that the antigenotoxic effects of p53 may be specific to deletions and complex-type mutations induced by double-strand breaks in DNA.
Environmental and Molecular Mutagenesis 04/2011; 52(3):244-52. · 3.71 Impact Factor
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Dongwei He,
Yoshihiko Uehara,
Maiko Furuya, Hironobu Ikehata,
Jun-ichiro Komura,
Kazumi Yamauchi,
Shizuko Kakinuma,
Yi Shang,
Yoshiya Shimada,
Akira Ootsuyama,
Toshiyuki Norimura,
Tetsuya Ono
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ABSTRACT: To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.
Mechanisms of ageing and development 02/2011; 132(3):117-22. · 4.18 Impact Factor
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ABSTRACT: Ultraviolet (UV) light induces specific mutations in the cellular and skin genome such as UV-signature and triplet mutations, the mechanism of which has been thought to involve translesion DNA synthesis (TLS) over UV-induced DNA base damage. Two models have been proposed: "error-free" bypass of deaminated cytosine-containing cyclobutane pyrimidine dimers (CPDs) by DNA polymerase η, and error-prone bypass of CPDs and other UV-induced photolesions by combinations of TLS and replicative DNA polymerases--the latter model has also been known as the two-step model, in which the cooperation of two (or more) DNA polymerases as misinserters and (mis)extenders is assumed. Daylight UV induces a characteristic UV-specific mutation, a UV-signature mutation occurring preferentially at methyl-CpG sites, which is also observed frequently after exposure to either UVB or UVA, but not to UVC. The wavelengths relevant to the mutation are so consistent with the composition of daylight UV that the mutation is called solar-UV signature, highlighting the importance of this type of mutation for creatures with the cytosine-methylated genome that are exposed to the sun in the natural environment. UVA has also been suggested to induce oxidative types of mutation, which would be caused by oxidative DNA damage produced through the oxidative stress after the irradiation. Indeed, UVA produces oxidative DNA damage not only in cells but also in skin, which, however, does not seem sufficient to induce mutations in the normal skin genome. In contrast, it has been demonstrated that UVA exclusively induces the solar-UV signature mutations in vivo through CPD formation.
Journal of Radiation Research 01/2011; 52(2):115-25. · 1.68 Impact Factor
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ABSTRACT: p53 suppresses the genomic instability provoked by genotoxic agents. Ultraviolet (UV) B induces skin cancers by producing DNA damage and mutations in the skin genome, whereas the skin tissue responds to the UVB insult with cell cycle arrest and apoptosis as well as damage exclusion by DNA repair. To address the p53 contribution to these skin responses in vivo, we analyzed the time course of DNA damage removal, apoptosis induction and hyperplasia in the skin after UVB irradiation in p53-knockout mice. We also examined UVB-induced mutations in the skin. We found that p53 deficiency does not abolish the UVB-induced apoptotic response in the epidermis but delays the process and the following hyperplasia 12-24 h. Regardless of the p53 genotype, 1 kJ/m(2) UVB induced a total replacement of the epidermal layer by destroying the damaged epidermis by apoptosis and rebuilding a new one through hyperplasia. We failed to detect a clear defect in removal of UVB-induced DNA photolesions from the genome of the p53-deficient skin except for a delay in the epidermis, which seemed to result from the delay in the apoptotic response. However, we found that p53 deficiency enhanced UVB-induced mutagenesis. Furthermore, in a genetic study using Xpa-knockout mice, we showed that the enhanced mutagenic response depends on the activity of nucleotide excision repair (NER), which was also supported by the mutation spectrum observed in the UVB-exposed p53-knockout mice. These results indicate that p53 protects the skin genome from the UVB genotoxicity by facilitating NER, whereas its contribution to the UVB-induced apoptosis is limited.
Mutagenesis 05/2010; 25(4):397-405. · 3.18 Impact Factor
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ABSTRACT: In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 08/2009; 670(1-2):24-31. · 2.85 Impact Factor
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Yoshihiko Uehara, Hironobu Ikehata,
Jun-ichiro Komura,
Ari Ito,
Masaki Ogata,
Tsunetoshi Itoh,
Ryoichi Hirayama,
Yoshiya Furusawa,
Koichi Ando,
Tatjana Paunesku,
Gayle E Woloschak,
Kenshi Komatsu,
Shinya Matsuura,
Tsuyoshi Ikura,
Kenji Kamiya,
Tetsuya Ono
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ABSTRACT: With the goal of understanding the role of non-homologous end-joining repair in the maintenance of genetic information at the tissue level, we studied mutations induced by radiation and subsequent repair of DNA double-strand breaks in Ku70 gene-deficient lacZ transgenic mice. The local mutation frequencies and types of mutations were analyzed on a lacZ gene that had been chromosomally integrated, which allowed us to monitor DNA sequence alterations within this 3.1-kbp region. The mutagenic process leading to the development of the most frequently observed small deletions in wild-type mice after exposure to 20 Gy of X rays was suppressed in Ku70(-/-) mice in the three tissues examined: spleen, liver and brain. Examination of DNA break rejoining and the phosphorylation of histone H2AX in Ku70-deficient and -proficient mice revealed that Ku70 deficiency decreased the frequency of DNA rejoining, suggesting that DNA rejoining is one of the causes of radiation-induced deletion mutations. Limited but statistically significant DNA rejoining was found in the liver and brain of Ku70-deficient mice 3.5 days after irradiation, showing the presence of a DNA double-strand break repair system other than non-homologous end joining. These data indicate a predominant role of non-homologous end joining in the production of radiation-induced mutations in vivo.
Radiation Research 09/2008; 170(2):216-23. · 2.68 Impact Factor
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Hironobu Ikehata,
Kazuaki Kawai,
Jun-ichiro Komura,
Ko Sakatsume,
Liangcheng Wang,
Masaru Imai,
Shoichi Higashi,
Osamu Nikaido,
Kazuo Yamamoto,
Kotaro Hieda,
Masakatsu Watanabe,
Hiroshi Kasai,
Tetsuya Ono
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ABSTRACT: UVA1 induces the formation of 8-hydroxy-2'-deoxyguanosines (8-OH-dGs) and cyclobutane pyrimidine dimers (CPDs) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA1 has not been clarified. We irradiated living mouse skin with 364-nm UVA1 laser light and analyzed the DNA damage formation and mutation induction in the epidermis and dermis. Although dose-dependent increases were observed for both 8-OH-dG and CPD, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C --> T transition at a dipyrimidine site. Moreover, these UV-specific mutations occurred preferentially at the 5'-TCG-3' sequence, suggesting that CpG methylation and photosensitization-mediated triplet energy transfer to thymine contribute to the CPD-mediated UVA1 genotoxicity. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA1 exposure. We also found differences in the responses to the UVA1 genotoxicity between the epidermis and the dermis: the mutation induction after UVA1 irradiation was suppressed in the dermis at all levels of irradiance examined, whereas it leveled off from a certain high irradiance in the epidermis.
Journal of Investigative Dermatology 03/2008; 128(9):2289-96. · 6.31 Impact Factor
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ABSTRACT: During mitosis in higher eukaryotic cells, transcription is silenced and transcription complexes are absent from promoters in the condensed chromosomes; however, epigenetic information concerning the pattern of expressed and silent genes must be preserved. Recently, it has been reported that CTCF, a major protein in vertebrate insulator elements, remains associated with mitotic chromatin. If the structure of insulators is preserved during mitosis, then it is possible that insulators can function as components or elements of the mechanism involved in the transfer of epigenetic information through the mitotic phase and can help guide the reconstitution of domain structure and nuclear organization after the completion of this phase. We have studied the chromatin structure of the insulator upstream of the c-MYC gene in mitotic HeLa cells. The region of the insulator corresponds to the DNase I hypersensitive site I, but Southern blot analysis revealed that hypersensitivity was lost during mitosis. High resolution in vivo footprinting analysis using dimethyl sulfate, UV light, psoralen, and DNase I also demonstrated the disappearance of the sequence-specific direct binding of CTCF and the absence of detectable structures during mitosis. Thus, it appears that the nucleoprotein complex involving this insulator element must be reassembled de novo with each new cell generation.
Proceedings of the National Academy of Sciences 11/2007; 104(40):15741-6. · 9.68 Impact Factor
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ABSTRACT: A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC-->TTT; Type IIa, NCC-->PyTT or PyCT (Py, pyrimidine); Type IIb, TCC-->PuTT or PuCT (Pu, purine); Type III, NCC-->NAT or NTA; Type IV, NTT-->AAT; Type Va, NCT-->NTX; and Type Vb, PuCT-->XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the 'A-rule', and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.
DNA Repair 05/2007; 6(5):658-68. · 4.14 Impact Factor
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ABSTRACT: Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.
Environmental and Molecular Mutagenesis 02/2007; 48(1):1-13. · 3.71 Impact Factor
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ABSTRACT: Mutations of the Xpc gene cause a deficiency in global genome repair, a subpathway of nucleotide excision repair (NER), in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of an Xpc null mutation (Xpc-/-) on damage induction, repair and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpc-/- and wild-type mice. CPDs were not significantly removed in either of the mouse genotypes by 12h after irradiation, whereas removal of 64PPs was observed in the wild-type. Irradiation with 300 and 400J/m2 UVB increased the lacZ mutant frequency in the Xpc-/- epidermis to at least twice as high as in the wild-type. Ninety-nine lacZ mutants isolated from the UVB-exposed epidermis of Xpc(-/-)mice were analyzed and compared with mutant sequences from irradiated wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in the dominance of C-->T transitions at dipyrimidine sites; however, Xpc-/- mice had a higher frequency of two-base tandem substitutions, including CC-->TT mutations, three-base tandem substitutions and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We concluded that the triplet mutation is a UV-specific mutation that preferably occurs in NER deficient genetic backgrounds.
DNA Repair 02/2007; 6(1):82-93. · 4.14 Impact Factor
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ABSTRACT: A C-terminal 183 amino acid-truncated mutation of the mouse Xpg gene (XpgDeltaex15) gives rise to a partial deficiency in nucleotide excision repair in homozygously affected cells. We studied the effect of this mutation on UVB-induced mutagenesis in mouse skin, using transgenic mice harboring lambda-phage-based bacterial lacZ genes as a mutational reporter. UVB increased the lacZ mutant frequency in the epidermis moderately in the homozygous mutant mice, but significantly higher than in the wild-type or the heterozygous mice, whereas background mutant frequencies were not appreciably different among the three mouse genotypes. Ninety-eight lacZ mutant sequences isolated from the UVB-exposed epidermis of the XpgDeltaex15-homozygous mice were analyzed and compared with mutant sequences from the wild-type mice. The spectra of the mutations in the two mouse genotypes were not significantly different, and they were highly UV-specific. There were frequent C --> T transitions at dipyrimidine sites and several CC --> TT tandem mutations, although the UV-specific mutations occurred more frequently at CpG sites in the mutant mice. The distribution of the mutations observed in the lacZ transgene and the preferred sequence context of the UV-specific C --> T mutations (5'-TC-3' > 5'-CC-3' > 5'-CT-3') in the Xpg-mutant mice were similar to those found in the wild-type mice. Despite these similarities, we detected a previously unrecognized type of the UV-induced mutation only in the Xpg mutant (6/98 in the mutation spectrum of the mutant vs. 0/76 in the wild-type; P = 0.035), which is characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We propose that this putative new class of mutation, which we refer to as a "triplet mutation", is characteristic of UV-induced mutation in an excision-repair-deficient background.
Environmental and Molecular Mutagenesis 04/2006; 47(2):107-16. · 3.71 Impact Factor
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ABSTRACT: We studied the mutations induced in skin by sunlight using transgenic Muta mice. Noon sunlight during summer at Sendai, Japan induced mutations efficiently in both epidermis and dermis. The mutant frequency (MF) in epidermis reached nearly 0.5% during the first 40 min irradiation but became saturated at this level with the appearance of skin inflammation after further irradiation. At the equivalent inflammatory dose, sunlight was twice as genotoxic as 313 nm-peak UVB. The 81 mutations detected in 80 lacZ transgene mutants isolated from the sunlight-exposed epidermis were dominated by C --> T transitions (89%), occurring exclusively at dipyrimidine sites, and also included a CC --> TT tandem substitution. Thus, the sunlight-induced mutation spectrum is highly UV-specific, quite similar to that induced by UVB but significantly different from that induced by UVA. Although oxidative damage-related C --> A transversions were detected only in five mutants (6%), their frequency was elevated to at least 15 times the background level, suggesting that the contribution of UVA-mediated oxidative stress is comparatively small but considerable. An analysis of bases adjacent to the mutated cytosines revealed that the sunlight-induced mutations prefer 5'-TC-3' dipyrimidine sites to 5'-CC-3' and 5'-CT-3'. The distribution of the frequent C --> T transition sites in the transgene was well associated with the CpG motif, which is known to be completely methylated in the gene, and quite similar to that induced by UVB rather than that by UVA. Thus, the UVB component contributes to the sunlight-induced mutations in the mammalian skin much more than the UVA component, whose influence through reactive oxygen species (ROS)-mediated mutagenesis is still appreciable.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 12/2004; 556(1-2):11-24. · 2.85 Impact Factor
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ABSTRACT: In an attempt to evaluate the possible role of mutations in the age-dependent increase of tumor incidence, we studied the mutational burden that accumulates in the aging process in different parts of the digestive tract in mice. The mutations were monitored in lacZ genes integrated in the mouse genome. The digestive tract was divided into the esophagus, stomach, proximal, medial, and distal part of the small intestine, and the colon. Epithelial tissues were separated from these tissues with the exception of the esophagus, in which case the whole tissue was examined. At a young age, the mutant frequencies as well as the molecular nature of the mutations were similar among the tissues examined. In old age, on the other hand, mutant frequencies were elevated to different degrees among the tissues; they were high in the small intestine and colon, intermediate in the stomach, and low in the esophagus. The molecular characteristics of the mutations also revealed distinct tissue-specificity; there were elevated rates of a small deletion mutation in the esophagus, G:C to T:A transversion in the proximal small intestine, and multiple mutations in the distal small intestine and colon. The results indicate that different parts of the digestive tract suffer from different kinds of mutational stress in the aging process. The nature of the multiple mutations suggests the presence of a mutator phenotype based on an imbalance in deoxyribonucleotide pools.
Cancer Research 11/2004; 64(19):6919-23. · 7.86 Impact Factor
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Tetsuya Ono,
Yusuke Saito,
Jun-Ichiro Komura, Hironobu Ikehata,
Yoshiaki Tarusawa,
Toshio Nojima,
Katsuo Goukon,
Yoshifumi Ohba,
Jianqing Wang,
Osamu Fujiwara,
Risaburo Sato
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ABSTRACT: A possible mutagenic effect of 2.45 GHz radiofrequency exposure was examined using lacZ-transgenic Muta mice. Pregnant animals were exposed intermittently at a whole-body averaged specific absorption rate of 0.71 W/kg (10 seconds on, 50 seconds off which is 4.3 W/kg during the 10 seconds exposure). Offspring that were exposed in utero for 16 hours a day, from the embryonic age of 0 to 15 days, were examined at 10 weeks of age. To minimize thermal effects, the exposure was given in repeated bursts of 10 seconds of exposure followed by 50 seconds of no exposure. Mutation frequencies at the lacZ gene in spleen, liver, brain, and testis were similar to those observed in non-exposed mice. Quality of mutation assessed by sequencing the nucleotides of mutant DNAs revealed no appreciable difference between exposed and non-exposed samples. The data suggest that the level of radiofrequency exposure studied is not mutagenic when administered in utero in short repeated bursts.
The Tohoku Journal of Experimental Medicine 03/2004; 202(2):93-103. · 1.24 Impact Factor
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ABSTRACT: We studied the kinetics of mutation induction in skin epidermis and dermis of UVA-irradiated transgenic Muta mice and analyzed the sequence changes in 80 lacZ transgene mutants from the irradiated epidermis. The mutant frequency increased linearly in both the epidermis and dermis up to 240 kJ/m2 UVA, twice as efficiently in the epidermis as in the dermis, without provoking any inflammatory reactions in the exposed skin. The 83 mutations detected in the UVA-exposed epidermis were dominated by C-->T transitions (88%), found almost exclusively at dipyrimidine sites, and specified by four occurrences of CC-->TT tandem substitutions, suggesting that UV-specific photoproducts induced in DNA have a major role in the genotoxicity. No T-->G transversions, which have been considered as a UVA signature mutation, and few mutations suggesting the relevance of oxidative damage were recovered in the present study. An analysis of the bases adjacent to the mutated cytosines revealed that the 3'-cytosine of dipyrimidine sites is the preferred target of UVA-induced C-->T transition. Moreover, C-->T transitions were induced at dipyrimidine sites associated with CpG much more frequently by UVA than by UVB, forming hotspots at several of these sites. These results suggest that UVA contributes more to the formation of recurrent or hotspot mutations at methylated CpG sites in the mammalian genome than UVB, since methylation of the CpG motif is observed entirely in the lacZ transgenes and is known to enhance the formation of cyclobutane pyrimidine dimers by longer wavelength UV.
Mutagenesis 11/2003; 18(6):511-9. · 3.18 Impact Factor
