Bipyrimidine photoproducts rather than oxidative lesions are the main type of DNA damage involved in the genotoxic effect of solar UVA radiation

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320-340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290-320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC-MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (lambda > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidine dimers (CPDs) are formed in a 1:1:3:10 ratio. In addition, we demonstrate that, in contrast to UVB and sunlight, UVA generates CPDs with a large predominance of TT CPDs, which strongly suggests that they are formed via a photosensitized triplet energy transfer. Moreover, UVA induces neither (6-4) photoproducts nor their Dewar isomers via direct absorption. We also show that UVA photons contained in sunlight, rather than UVB, are implicated in the photoisomerization of (6-4) photoproducts, a quickly repaired damage, into poorly repaired and highly mutagenic Dewar photoproducts. Altogether, our data shed new light on the deleterious effect of UVA.     

The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli

Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV-induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV-induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light-induced mutations such as deletions. None of the available evidence rules out (6-4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutagenic DNA repair in Escherichia coli. XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage.

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

The Role of Pyrimidine Dimers as Premutagenic Lesions: A Study of Targeted VS. Untargeted Mutagenesis in the lacI Gene of ESCHERICHIA COLI

We have employed conjugal transfer of an F' lac episome to examine targeted and untargeted mutagenesis in the lacI gene of Escherichia coli and to determine the relative importance of pyrimidine dimers as premutational UV lesions compared to (6-4) photoproducts that also may have a mutational role. This conjugal system allowed us to assess the premutagenic role of UV lesions independently from any role as inducers of SOS functions. F' DNA was transferred to an SOS-induced recipient strain from: unirradiated donor cells, UV-treated donor cells or donor cells that were irradiated and then exposed to photoreactivating light. The results indicate that SOS-related, untargeted events may account for as much as one-third of the nonsense mutations (i.e., base substitutions) recovered after undamaged F' DNA is transferred to UV-irradiated recipients. When the donor strain also is irradiated, in excess of 90% of the mutations detected following conjugation appear to be targeted. Photoreactivation of the UV-treated donors cells, prior to F' transfer to the SOS-induced recipient strain, demonstrated that in this experimental system virtually all recovered UV-induced mutations are targeted by photoreactivable lesions. We presume that these lesions are pyrimidine dimers because (6-4) photoproducts are not photoreactivable.     

Mutations induced by ultraviolet light

The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA bases. We also discuss the role of DNA damage-tolerant DNA polymerases in UV lesion bypass and mutagenesis.     

Thermal resistance to photoreactivation of ultraviolet light induced mutations in the lacI gene of E. coli ung

Ultraviolet light (UV) induced mutations in the lacI gene of Escherichia coli are thought to be targeted by DNA photoproducts. A number of reports suggest that both cyclobutyl pyrimidine dimers and pyrimidine (6−4) pyrimidone photoproducts may be involved. To investigate the potential contribution of each of these DNA photoproducts to mutagenesis in the lacI gene, we held UV-irradiated cells at a temperature of 44°C for 75 min and then exposed them to photoreactivating light (PR). This protocol is expected to preferentially deaminate specifically those cytosines that are contained in cyclobutyl dimers and subsequently monomerize the dimers to yield uracils in the DNA. In a strain deficient for uracil-DNA glycosylase (Ung⁻), these uracils would not be removed and a G : C → A : T transition would result at the site of the dimer. This protocol resulted in the enhancement of amber nonsense mutations that result from transitions at potential cytosine-containing dimer sites. The enhanced mutation frequencies resulting from this procedure were used to estimate the probability of dimer formation at the individual sites. A comparison of the dimer distribution with the mutation frequencies following UV alone suggests that both cyclobutyl dimers and (6−4) photoproducts contribute to UV-mutagenesis in the lacI gene. In addition, we conclude that the frequency of mutation at any particular site not only reflects the occurrence of DNA damage, but also the action of metabolic processes that are responsible for DNA repair and mutagenesis.     

Mutagenic DNA repair in Escherichia coli XXI. A stable SOS-inducing signal persisting after excision repair of ultraviolet damage

Mutations to streptomycin resistance induced by ultraviolet light in Escherichia coli can lose their susceptibility to photoreversing light during excision repair and in the absence of chromosomal replication and protein synthesis, i.e., under conditions where SOS induction cannot occur. Using fusions of lac with sulA and umuC we have shown that after excision of UV damage in the presence of chloramphenicol there is a persisting, relatively stable signal capable of inducing SOS genes when protein sysnthesis is subsequently permitted. The persisting signal is formed roughly in proportion to the square of the UV dose and is about 30% photoreversible. It is suggested that the persisting SOS-inducing signal comprises a UV photoproduct (the target lesion) opposite a gap in the opposing DNA strand, and is formed by excision of one (the ancillary lesion) of a pair of closely opposed photoproducts. Calculations suggest that as few as two or three such configurations in a cell can lead to induction a sulA when protein synthesis is permitted. It is not clear whether these configurations can directly induce the SOS system because of their region of single-stranded DNA or whether the ultimate SOS-inducing signal is a more extensive single-stranded region formed when such configurations encounter a replication fork. Photoproduct/gap configurations have been previously suggested to be potentially mutagenic. UV-induced mutations to streptomycin resistance are mostly at A:T sites and are not photoreversible in fully SOS-induced bacteria in the absence of excision repair, indicating that they are not targeted at cyclobutane-type pyrimidine dimers. In SOS-induced excision-proficient bacteria there is about 39% photoreversibility which is rapidly lost after UV. This photoreversibility is attributed to many ancillary lesions being cyclobutane-type pyrimidine dimers which are excised leading to the exposure of target lesions on the opposing strand which, at these particular sites, are mostly non-photoreversible photoproducts.     

Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light

In targeted mutagenesis of lambda phage by ultraviolet light, the mutations are caused by radiation-induced lesions in the phage DNA. Of 62 mutations in the lambda cI gene that were sequenced, 41 (63%) of the targeted mutations were transitions, with similar numbers of C X G to T X A and T X A to C X G base changes. The remaining 21 mutations were about equally divided among eight transversions, seven frameshifts (5 additions and 2 deletions), and six double events with either two nearby base changes or a base change and a nearby frameshift. Of the 62 mutations, 60 could be associated with -Pyr-Pyr- sequences in the DNA, sites of likely photoproducts. For more information on this point, lambda phage were irradiated with 313 nm light in the presence of acetophenone, for which the major photoproduct is reported to be the thymine-thymine cyclobutyl dimer, with no measurable Pyr(6-4)Pyo photoproducts. Of 22 mutations sequenced, 19 were transversions and only one was a transition, permitting the conclusion that thymine-thymine cyclobutyl dimers are not the primary cause of ultraviolet light-induced transitions. A consideration of all the data strongly suggests that Pyr(6-4)Pyo photoproducts are mutagenic lesions.     

Mutability of bacteriophage M13 by ultraviolet light: Role of pyrimidine dimers

Summary The role of pyrimidine dimers in mutagenesis by ultraviolet light was examined by measuring the UV-induced reversion of six different bacteriophage M13 amber mutants for which the neighboring DNA sequences are known. The mutational response at amber (TAG) codons preceded by a guanine or adenine (where no pyrimidine dimer can be formed) were compared with those preceded by thymine or cytosine (where dimer formation is possible). Equivalent levels of UV-induced mutagenesis were observed at both kinds of sites. This observation demonstrates that there is no requirement for a pyrimidine dimer directly at the site of UV-induced mutation in this single-stranded DNA phage. UV irradiation of the phage was also performed in the presence of Ag⁺ ions, which specifically sensitize the DNA to dimer formation. The two methods of irradiation, when compared at equal survival levels (and presumably equal dimer frequencies), produced equivalent frequencies of reversion of the amber phage. We believe these results indicate that while the presence of pyrimidine dimers may be a prerequisite for UV mutagenesis, the actual mutagenic event can occur at a site some distance removed from a dimer.     

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.     

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.     

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.     

Similar mutagenicity of photoactivated porphyrins and ultraviolet A radiation in mouse embryonic fibroblasts: involvement of oxidative DNA lesions in mutagenesis

Ultraviolet A (UVA) radiation is implicated in the etiology of human skin cancer. However, the underlying mechanism of carcinogenicity for UVA is not fully delineated. A mutagenic role for UVA has been suggested, which involves activation of endogenous photosensitizers generating oxidative DNA damage. We investigated the mutagenicity of UVA alone and in combination with delta-aminolevulinic acid (delta-ALA), a precursor of the intracellular photosensitizers porphyrins, in transgenic Big Blue mouse embryonic fibroblasts. A significant generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a typical promutagenic oxidative DNA lesion, was observed in cells treated with a combination of delta-ALA (1 mM) and UVA (0.06 J/cm(2)) as quantified by high-pressure liquid chromatography-tandem mass spectrometry (p < 0.001; relative to the control). The steady-state level of 8-oxo-dG, however, remained unchanged in cells irradiated with UVA or treated with delta-ALA alone. Other photolesions including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were not detectable in cells treated with delta-ALA and/or irradiated with UVA as determined by terminal transferase-dependent polymerase chain reaction assay. Mutation analyses of the cII transgene in cells treated with a combination of delta-ALA and UVA showed an approximately 3-fold increase in mutant frequency relative to the control (p < 0.008), as well as a unique induced mutation spectrum as established by DNA sequence analysis (p < 0.005; 95% CI, 0.002-0.009). No mutagenic effects were observed in cells irradiated with UVA or treated with delta-ALA alone. The spectrum of mutations produced by delta-ALA plus UVA was characterized by a significantly increased frequency of G --> T transversions (p < 0.0003; relative to the control), which are the hallmark mutations induced by 8-oxo-dG. Notably, the 8-oxo-dG-mediated mutagenicity of UVA plus delta-ALA is similar to that established previously for UVA alone at a mutagenic dose of 18 J/cm(2). We conclude that, in the presence of exogenous photosensitizers, UVA at a nonmutagenic dose induces mutations through the same mechanism as does a mutagenic dose of UVA per se.     

Thermodynamics and kinetics for base pair opening in the DNA decamer duplexes containing cyclobutane pyrimidine dimer

The cyclobutane pyrimidine dimer (CPD) is one of the major classes of cytotoxic and carcinogenic DNA photoproducts induced by UV light. Hydrogen exchange rates of the imino protons were measured for various CPD-containing DNA duplexes to better understand the mechanism for CPD recognition by XPC-hHR23B. The results here revealed that double T·G mismatches in a CPD lesion significantly destabilized six consecutive base pairs compared to other DNA duplexes. This flexibility in a DNA duplex caused at the CPD lesions with double T·G mismatches might be the key factor for damage recognition by XPC-hHR23B.     

50 years thymine dimer

Fifty years ago thymine dimer was discovered in the Biochemical and Biophysical Laboratory of Delft Technological University, The Netherlands, by one of the authors of this review (Beukers) as the first environmentally induced DNA lesion. It is one of the photoproducts formed between adjacent pyrimidine bases in DNA by UV irradiation, currently known as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Major lesions found in DNA after in vitro or in vivo UV irradiation are the cis-syn cyclobutane thymine dimer and the thymine-cytosine (6-4) photoproduct. Even after 50 years the study of photo-induced DNA lesions is still going on as is illustrated by the hundreds of papers published every year and the millions hits when browsing the internet for dimer-related information. Living organisms possess efficient and different mechanisms to repair detrimental lesions in their DNA. A unique mechanism to repair CPDs is reversion by either direct interaction with light of short wavelength or by enzymatic photoreactivation. Photophysical mechanisms that induce and reverse molecular bonds in biological macromolecules have been a main focus of research of the group in Delft in the middle of the last century. This review describes the break-through results of these studies which were the result of intense interactions between scientists in the fields of physics, organic chemistry and biochemistry. Philosophically, the "view" of the group in Delft was very appealing: since in nature photolesions are induced in DNA by the sun, how is it possible that repair of these lesions could be accomplished by the same energy source. Evolutionary, it is hardly possible to think of a more efficient repair mechanism.