DNA strand specificity for UV-induced mutations in mammalian cells (original) (raw)
Related papers
Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster HPRT gene
Nucleic Acids Research, 1991
DNA excision repair modulates the mutagenic effect of many genotoxic agents. The recently observed strand specificity for removal of UV-induced cyclobutane dimers from actively transcribed genes in mammalian cells could influence the nature and distribution of mutations in a particular gene. To investigate this, we have analyzed UV-induced DNA repair and mutagenesis in the same gene, i.e. the hypoxanthine phosphoribosyl-transferase (hprt) gene. In 23 hprt mutants from V79 Chinese hamster cells induced by 2 J/m2 UV we found a strong strand bias for mutation induction: assuming that pre-mutagenic lesions occur at dipyrimidine sequences, 85% of the mutations could be attributed to lesions in the nontranscribed strand. Analysis of DNA repair in the hprt gene revealed that more than 90% of the cyclobutane dimers were removed from the transcribed strand within 8 hours after irradiation with 10 J/m2 UV, whereas virtually no dimer removal could be detected from the nontranscribed strand even up to 24 hr after UV. These data present the first proof that strand specific repair of DNA lesions in an expressed mammalian gene is associated with a strand specificity for mutation induction.
Photochemistry and Photobiology, 2005
We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81 % of UV-A-induced early mutations (Le. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.
Photochemistry and Photobiology, 2004
We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81 % of UV-A-induced early mutations (Le. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.
Mutation research, 1992
A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-trans...
Proceedings of the …, 1987
The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. AU transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of >99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosinecontaining non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.
Characterization of mutations induced by 300 and 320 nm UV radiation in a rat fibroblast cell line
Mutation research, 1996
The cytotoxic and mutagenic activities of monochromatic ultraviolet light (UV) at four wavelengths (254, 290, 300 and 320 nm) were determined using a rat fibroblast cell line CREF stably infected with a retroviral vector carrying the neo and HSV-tk markers. In this system, mutations can be positively detected as acyclovir-resistant colonies. Although the action spectra for these activities closely fit some of the previously reported spectra for photochemical DNA modifications, erythema, cell killing and mouse skin carcinogenesis, they diverge at 320 nm from the absorption spectrum for DNA and the action spectrum for bacterial inactivation and mutagenesis. Structural comparison of the HSV-tk mutants detected after irradiation with 300 and 320 nm UV revealed (1) CC dimers and C oligomers as predominant targets at both wavelengths; (2) increased incidence of relatively large deletions at 300 nm; and (3) greatly increased frequency of tandem double mutations at both wavelengths and of c...
Molecular and cellular biology, 1988
Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189). We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation. Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced. In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm. These hot spots are shown to arise predominantly at sites of mutations involving mu...
Proceedings of the National Academy of Sciences, 1981
The distribution of UV light-induced damage to the highly reiterated ca sequence ofhuman DNA was investigated. The results show that the distribution of UV light-induced cyclobutane dimers within a defined sequence is similar whether the DNA is exposed to UV light as part of the chromosome of intact cells or as naked DNA. However, the cellular environment shields the nuclear DNA, resulting in about 50% decrease in apparent dose. A new type of UV photodamage was detected. Treatment of UV light-irradiated DNA with hot alkali results in strand breaks at positions of cytidine located 3' to pyrimidine nucleosides. The chemical nature and biological significance of the pyrimidine nucleoside-cytidine lesion is discussed. The UV portion of the spectrum of the sun induces damage to cellular DNA. In humans such damage can result in cell death or in precancerous lesions in exposed areas of the skin (1). UV irradiation results in similar phenomena in cultured human cells, causing cell death, mutation, and transformation to a malignant phenotype (2, 3). The UV light-induced lesion that has been studied in most detail is the cyclobutane pyrimidine dimer formed between adjacent pyrimidine bases to produce thymine-thymine, cytosine-thymine, and cytosine-cytosine dimers. Attempts to understand the biological effects of UV light have for the most part focused on the enzymatic repair of the pyrimidine photodimers. Here we investigate the distribution of UV light-induced damage in a defined sequence of human DNA. Use of the highly reiterated a DNA sequence (4-8) permits direct comparison of sites of DNA modification within a defined DNA sequence, when either purified DNA or intact human cells are exposed to light. In the course of these experiments, we discovered another major type of photodamage to DNA that may be of biological consequence.
UVB and UVA radiation-mediated damage to isolated and cellular DNA
Pure and Applied Chemistry, 2000
The effects of solar light on cellular DNA are mostly explained by both direct excitation of nucleobases and photosensitized reactions that are mediated by UVB and UVA radiation, respectively. A large body of information is now available on the main photodynamic reactions to DNA, which involve guanine as the preferential target of both one-electron oxidation and singlet oxygen oxidation, as the result of type I and type II mechanisms. Most of the final products of the photosensitized reactions of guanine base in isolated DNA have been characterized, and comprehensive mechanisms for their formation have been proposed. Further insights into the mechanisms of solar radiation-induced modifications within cellular DNA have been gained from accurate measurement of the main photoproducts using recently designed sensitive chromatographic and biochemical methods. Thus, the distribution pattern of the 12 possible bipyrimidine photoproducts has been shown to be similar in the DNA of UVB-irradiated rodent and human cells. Cyclobutyl pyrimidine dimers are also generated at di-thymine and thymine-cytosine sites within nuclear DNA upon exposure to UVA radiation as the likely result of triplet energy transfer. In addition, oxidative reactions that involved mostly singlet oxygen, and to a lesser extent, ؒ OH radicals are also implicated, although less efficiently. presentations are published in this issue, pp. 925-1085. ‡ Corresponding author pendent is the Paterno-Büchi photoreaction. This involves the transient formation of highly unstable oxetanes or azetidines that implicates a singlet excited-state pyrimidine and that leads to the generation of pyrimidine(6-4)pyrimidone photoadducts (6-4PPs). The latter photoproducts that exhibit strong absorption features in the near-UV range may be converted into the related Dewar valence isomers (DewPPs) through secondary photochemical processes at biological relevant doses. Four main bipyrimidine sites that involve thymine and/or cytosine are present in DNA. Therefore, 12 possible photoproducts, most of them having been characterized in numerous model studies, are expected to be generated in UVB-irradiated isolated or cellular DNA [2,3]. Until recently, only a paucity of information was available on the UVB-induced formation of each of the pyrimidine photoproducts in DNA due to the lack of appropriate methods.