UV-induced Mutation Spectra Research Articles

Biochemical and photochemical mechanisms that produce different UV-induced mutation spectra.

Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.

Significance of CpG Methylation for Solar UV‐Induced Mutagenesis and Carcinogenesis in Skin‡

Mutations detected in the p53 gene in human nonmelanoma skin cancers show a highly UV-specific mutation pattern, a dominance of C --> T base substitutions at dipyrimidine sites plus frequent CC --> TT tandem substitutions, indicating a major involvement of solar UV in the skin carcinogenesis. These mutations also have another important characteristic of frequent occurrences at CpG dinucleotide sites, some of which actually show prominent hotspots in the p53 gene. Although mammalian solar UV-induced mutation spectra were studied intensively in the aprt gene using rodent cultured cells and the UV-specific mutation pattern was confirmed, the second characteristic of the p53 mutations in human skin cancers had not been reproduced. However, studies with transgenic mouse systems developed thereafter for mutation research, which harbor methyl CpG-abundant transgenes as mutation markers, yielded complete reproductions of the situation of the human skin cancer mutations in terms of both the UV-specific pattern and the frequent occurrence at CpG sites. In this review, we evaluate the significance of the CpG methylation for solar UV mutagenesis in the mammalian genome, which would lead to skin carcinogenesis. We propose that the UV-specific mutations at methylated CpG sites, C --> T transitions at methyl CpG-associated dipyrimidine sites, are a solar UV-specific mutation signature, and have estimated the wavelength range effective for the solar-UV-specific mutation as 310-340 nm. We also recommend the use of methyl CpG-enriched sequences as mutational targets for studies on solar-UV genotoxicity for human, rather than conventional mammalian mutational marker genes such as the aprt and hprt genes.

Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase η from Arabidopsis thaliana

DNA polymerase η belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis– syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Polη homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Polη activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.

Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis.

The recently discovered human enzyme DNA polymerase iota (pol iota) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol iota in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol iota expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol iota expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol iota expression is an early event after UV-mediated DNA damage. That pol iota may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol iota was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that > 90% were point mutations. The analysis also demonstrated a decreased C --> T nucleotide transition and an increased C --> A transversion rate. Overall, our data strongly suggest that pol iota may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.

In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli

In vivo formation and repair of the major UV-induced DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs), have been examined at the gene and nucleotide level in Escherichia coli. Each type of DNA photoproduct has individually been studied using photoreactivation and two newly developed assays; the multiplex QPCR assay for damage detection at the gene level and the reiterative primer extension (PE) assay for damage detection at the nucleotide level. In the E. coli lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, during UV irradiation. Repair of 6-4 PPs is more efficient than repair of CPDs since, on the average, 42% of 6-4 PPs are repaired in both genes in the first 40 min following 200 J/m 2 UV irradiation, while 1% of CPDs are repaired. The location, relative frequency of formation, and efficiency of repair of each type of photoproduct was examined in the first 52 codons of the E. coli lacI gene at the nucleotide level. Hotspots of formation were found for each type of lesion. Most photoproducts are at sites where both CPDs and 6-4 PPs are formed. Allowing 40 min of recovery following 200 J/m 2 shows that in vivo repair of 6-4 PPs is about fourfold more efficient than the repair of CPDs. Comparison of the lesion-specific photoproduct distribution of the lacI gene with a UV-induced mutation spectrum from wild-type cells shows that most mutational hotspots are correlated with sites of a majority of CPD formation. However, 6-4 PPs are also formed at some of these sites with relatively high frequency. This information, taken together with the observation that 6-4 PPs are repaired faster than CPDs, suggest that the cause of mutagenic hotspots in wild-type E. coli is inefficient repair of CPDs.

Transcription-modulated repair in Escherichia coli evident with UV-induced mutation spectra in supF

We have determined several mutation spectra with the supF sequence after UV mutagenesis in Escherichia coli. The cells were either mfd+ or mfd− and grown in defined or complex medium. The tRNA supF gene was expressed from the plasmid pZ189 or pLS1D (similar to pLS189, a variant of pZ189, but with a tac promoter for supF). Most of the mutations with either plasmid could be attributed to possible targeting photoproducts at dipyrimidine sites in the transcribed (TS) or non-transcribed (NTS) DNA strand with differential characteristics relevant to the repair process “mutation frequency decline” (MFD): (1) with pZ189, targeting sites in TS were favored over sites in NTS in all conditions except after an explicit MFD incubation with mfd+ cells, when there was a majority in NTS; (2) with pLS1D (tac promoter), there was always a marked bias for targeting sites in TS and this was not altered by an MFD incubation; and (3) with pLS1D, spectra withmfd− cells vis-à-vis wild-type indicated a notable shift in the position of a hot-spot (both targeting sites in TS) and an increase in deletion mutations. The results support the Selby-Sancar idea that transcription-coupled nucleotide excision repair (TCR) at tRNA genes accounts for MFD and can be inhibited by rapid transcription. During interference of TCR by rapid transcription, however, the presence or absence of functional Mfd protein (transcription-repair coupling factor) can still influence the pattern of mutation, e.g. alter the position of a hot-spot in pLS1D. Only when a tRNA promoter is modulated by an MFD condition is transcription at a rate conducive to TCR. There were several deletion mutations with pLS1D between direct repeats (not present in pZ189) and a model for their production by UV damage is suggested. The spectra with pZ189 in E. coli had similarities with those published for UV mutagenesis in human cells, e.g. mutations at positions ∼124 and 156.

UVB-induced mutagenesis in hairless lambda lacZ-transgenic mice.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (MutaMouse), which contain the lambda gt10lacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m2 UVB or to two successive irradiations of either 200 and 800 J/m2 UVB, with intervals of 1, 3, or 5 days, or to 800 and 200 J/m2 UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. The mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C --> A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C --> A:T transitions at CpG sites. The results indicate that the hairless lambda lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations.

Use of an infectious Simian virus 40-based shuttle vector to analyse UV-induced mutagenesis in monkey cells

SV40 based shuttle vectors able to be packaged as pseudovirions have been used either as naked DNA or as pseudovirus to analyse the mutation frequency and the UV-induced mutation spectra obtained after transfection or infection of COS7 monkey cells. The frequency of supF spontaneous mutants was similar whatever the state of the vector, indicating that the transfection step is not responsible for the high spontaneous mutation frequency when using shuttle vectors. Nevertheless the UV-induced mutation frequency of the supF gene was higher when transfected DNA was replicated into COS7 cells than when pseudovirus infection was performed. The UV induced mutation spectra was basically similar in both situations but a new hot-spot at nucleotide 110 was obtained after pseudovirus infection. UV-pretreated and control COS7 cells were infected with untreated or UV-damaged πSVPC7 shuttle virus and the survival and the supF mutation frequency were analysed in the progeny. The survival of UV-damaged pseudovirus replicated in 10 J/m2 UV-pretreated cells was 2-fold higher than in untreated cells. This increase in the survival was accompanied by a slight enhancement in the number of supF− mutants.

Lessons learned from xeroderma pigmentosum.

Lessons learned from xeroderma pigmentosum.

High Resolution Mapping of UV-induced Photoproducts in the Escherichia coli lacI Gene: Inefficient Repair of the Non-transcribed Strand Correlates with High Mutation Frequency

UV-induced DNA photoproduct formation and repair has been examined at the gene and nucleotide level in Escherichia coli using two newly developed quantitative assays. A multiplex quantitative PCR assay was used to measure photoproduct formation and repair at the gene level in both the constitutive lacI gene and the inducible lacZ gene, simultaneously. Both genes displayed similar photoproduct formation frequencies (0·4 lesions/kb per 100 J/m2). Following a 15 minute recovery period, 36% and 39% of the damage resulting from 100 J/m2 was removed from the lacI and lacZ genes, respectively. Under the growth conditions applied, the lacZ gene was expressed at a very low rate resulting in 0·3% of β-galactosidase activity as compared to induced cells. A newly developed reiterative primer extension assay has been employed to examine photoproduct formation and repair at the nucleotide level. Analysis of UV-induced DNA photoproducts in the first 184 base-pairs of the lacI gene of genomic E. coli DNA has revealed that photoproducts are induced linearly with dose and the slope is sequence context-dependent. A post-irradiation recovery period revealed differences in the repair efficiency at individual nucleotides. Repair of photoproducts on the transcribed strand was generally twice as efficient as repair of photoproducts on the non-transcribed strand, indicating the strand-specific DNA repair occurs in the constitutively transcribed lacI gene of E. coli. Comparison of the UV-induced DNA photoproduct distribution with an established UV-induced mutation spectrum from wild-type cells revealed that photoproducts form at all mutagenic hotspots. Some sites of low frequency mutations were not observed to be sites of photoproduct formation. However, not all photoproducts appeared to be mutagenic. This was especially true for those on the efficiently repaired transcribed strand. It is hypothesized that the preferential repair of photoproducts on this strand may prevent may of these photoproduct sites from becoming mutagenic hotspots. These data strongly support the hypothesis that mutations arise at inefficiently repaired photoproducts on the non-transcribed strand.

Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients.

The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. We have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are > 100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze > 40 XP skin tumors (mainly basal and squamous cell carcinomas), we have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC-->TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues.