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.     

Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells. Possible role of Dewar photoproducts in solar mutagenesis

In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.     

UV-targeted dinucleotides are not depleted in light-exposed prokaryotic genomes

We have investigated the hypothesis that pyrimidine dinucleotides are avoided in light-exposed genomes as the result of selective pressure due to high ultraviolet (UV) exposure. The main damage to DNA produced by UV radiation is known to be the formation of pyrimidine photoproducts: it is estimated that about 10 dimers per minute are formed in an Escherichia coli chromosome exposed to the UV light in direct overhead sunlight at sea level. It is also known that on an E. coli chromosome exposed to UVb wavelengths (290-320 nm), pyrimidine photoproducts are formed in the following proportions: 59% TpT, 7% CpC, and 34% CpT plus TpC. We have analyzed all available complete prokaryotic genomes and the model organism Prochlorococcus marinus and have found that pyrimidine dinucleotides are not systematically avoided. This suggests that prokaryotes must have sufficiently effective protection and repair systems for UV exposure to not affect their dinucleotide composition.     

Characterization of DNA repair in a mutant cell line derived from ICR 2A frog cells that is hypersensitive to non-dimer DNA damages induced by solar ultraviolet radiation

The level of excision repair and the inhibition and recovery of semiconservative DNA synthesis were examined following the induction of non-dimer DNA damages by solar ultraviolet radiation in a mutant cell line, DRP 36, derived from ICR 2A frog cells that is hypersensitive to these lesions. A relatively pure population of non-dimer photoproducts was produced by exposure of cells to the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp followed by treatment with photoreactivating light (PRL) which removes most of the small yield of dimers induced by the irradiation. Using a modification of the bromodeoxyuridine (BrdUrd) photolysis assay, that enhances the sensitivity of this assay, it was found that DRP 36 cells perform a significantly lower level of excision repair following the induction of non-dimer DNA damages compared with the ICR 2A cells. In contrast, the level of excision repair of 254-nm-induced dimers was similar in the two cell lines. In addition, the induction of non-dimer damages caused a greater inhibition of DNA synthesis that persisted for a longer period of time in the mutant compared with the parental cells. Hence, these results indicate that the DRP 36 cells are deficient in the repair of at least one type of solar UV-induced non-dimer lesion.     

Quantitation and Visualization of Ultraviolet‐Induced DNA Damage Using Specific Antibodies: Application to Pigment Cell Biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6‐4)photoproducts (6‐4PPs) and Dewar isomers of 6‐4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6‐4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme‐linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser‐scanning confocal microscope. This latter method allows us to reconstruct three‐dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

Formation of the main UV-induced thymine dimeric lesions within isolated and cellular DNA as measured by high performance liquid chromatography-tandem mass spectrometry

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.     

Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. Dimers were induced by exposure of cells to 254 nm uv, while nondimer photoproducts were induced by irradiation of cells with uv produced by a fluorescent sunlamp (FSL) that was filtered through 48A Mylar (removes wavelengths less than 310 nm). The FSL-irradiated cultures were also treated with photoreactivating light (PRL) which removed most of the small number of dimers induced by the irradiation, leaving a relatively pure population of nondimer photoproducts. In addition, cells were exposed to 60Co gamma rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in gamma-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.     

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast.

A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.     

The two major spore DNA repair pathways, nucleotide excision repair and spore photoproduct lyase, are sufficient for the resistance of Bacillus subtilis spores to artificial UV-C and UV-B but not to solar radiation.

Bacterial endospores are 1 to 2 orders of magnitude more resistant to 254-nm UV (UV-C) radiation than are exponentially growing cells of the same strain. This high UV resistance is due to two related phenomena: (i) DNA of dormant spores irradiated with 254-nm UV accumulates mainly a unique thymine dimer called the spore photoproduct (SP), and (ii) SP is corrected during spore germination by two major DNA repair pathways, nucleotide excision repair (NER) and an SP-specific enzyme called SP lyase. To date, it has been assumed that these two factors also account for resistance of bacterial spores to solar UV in the environment, despite the fact that sunlight at the Earth's surface consists of UV-B, UV-A, visible, and infrared wavelengths of approximately 290 nm and longer. To test this assumption, isogenic strains of Bacillus subtilis lacking either the NER or SP lyase DNA repair pathway were assayed for their relative resistance to radiation at a number of UV wavelengths, including UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight, and sunlight from which the UV-B portion had been removed. For purposes of direct comparison, spore UV resistance levels were determined with respect to a calibrated biological dosimeter consisting of a mixture of wild-type spores and spores lacking both DNA repair systems. It was observed that the relative contributions of the two pathways to spore UV resistance change depending on the UV wavelengths used in a manner suggesting that spores irradiated with light at environmentally relevant UV wavelengths may accumulate significant amounts of one or more DNA photoproducts in addition to SP. Furthermore, it was noted that upon exposure to increasing wavelengths, wild-type spores decreased in their UV resistance from 33-fold (UV-C) to 12-fold (UV-B plus UV-A sunlight) to 6-fold (UV-A sunlight alone) more resistant than mutants lacking both DNA repair systems, suggesting that at increasing solar UV wavelengths, spores are inactivated either by DNA damage not reparable by the NER or SP lyase system, damage caused to photosensitive molecules other than DNA, or both.     

Photoprotection of DNA (in vitro) by acyclothymidine dinucleosides

Excessive exposure to sunlight is primarily implicated in ultraviolet (UV) induced skin cancers worldwide. Direct absorption of UV radiation by DNA leads to the formation of cyclobutane pyrimidine dimers (CPDs) resulting in DNA damage. The molecular mechanisms involved in the mutagenicity of CPDs are well established. Photoprotection of the skin from the detrimental effects of UV is essential in preventing skin damage. A variety of formulations, which essentially contain UV filters have been used as photoprotective agents of the skin. These comprise aromatic and inorganic molecules, whose mechanism of action involves either absorption, reflection, or scattering of UV radiation. However, the downstream photoproducts of some of these molecules have undesirable characteristics which compromise their utility. A biomimetic approach involving structural analogs of nucleic acids can help overcome these limitations. Herein, we show the photoprotective action of acyclothymidine dinucleosides on both plasmid and cellular DNA.     

Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA.

Hot piperidine is often used to cleave abasic and UV-irradiated DNA at the sites of damage. It can inflict non-specific damage on DNA, probably because it is a strong base and creates significant concentrations of hydroxyl ions which can attack purines and pyrimidines. We show that several other amines can cleave abasic DNA at or near neutral pH without non-specific damage. One diamine, N,N'-dimethylethylenediamine, efficiently cleaves abasic DNA at pH 7.4 by either beta- or beta,delta-elimination, depending on temperature. Using end-labelled oligonucleotides we show that cleavage depends mainly on elimination reactions, but that 4',5'-cyclization is also significant. This reagent also cleaves at photoproducts induced by UVC and UVB, producing the same overall pattern as piperidine, but with no non-specific damage. It should prove valuable in locating low levels of photoproducts in DNA, such as those induced by natural sunlight.     

Differential repair of UVB-induced cyclobutane pyrimidine dimers in cultured human skin cells and whole human skin

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are the two main classes of mutagenic DNA damages induced by UVB radiation. Numerous studies have been devoted so far to their formation and repair in human cells and skin. However, the biochemical methods used often lack the specificity that would allow the individual study of each of the four CPDs and 6-4PPs produced at TT, TC, CT and CC dinucleotides. In the present work, we applied an HPLC-mass spectrometry assay to study the formation and repair of CPDs and 6-4PPs photoproducts in primary cultures of human keratinocytes and fibroblasts as well as in whole human skin. We first observed that the yield of dimeric lesions was slightly higher in fibroblasts than in keratinocytes. In contrast, the rate of global repair was higher in the last cell type. Moreover, removal of DNA photoproducts in skin biopsies was found to be slower than in both cultured skin cells. In agreement with previous works, the repair of 6-4PPs was found to be more efficient than that of CPDs in the three types of samples, with no observed difference between the removal of the TT and TC derivatives. In contrast, a significant influence of the nature of the two modified pyrimidines was observed on the repair rate of CPDs. The decreasing order of removal efficiency was the following: C<>T>C<>C>T<>C>T<>T. These data, together with the known intrinsic mutational properties of the lesions, would support the reported UV mutation spectra. A noticeable exception concerns CC dinucleotides that are mutational hotspots with an UV-specific CC to TT tandem mutation, although related bipyrimidine photoproducts are produced in low yields and efficiently repaired.     

Effect of the GC content of DNA on the distribution of UVB-induced bipyrimidine photoproducts.

Solar UV radiation is a major mutagen that damages DNA through the formation of dimeric photoproducts between adjacent thymine and cytosine bases. A major effect of the GC content of the genome is thus anticipated, in particular in prokaryotes where this parameter significantly varies among species. We quantified the formation of UV-induced photolesions within both isolated and cellular DNA of bacteria of different GC content. First, we could unambiguously show the favored formation of cytosine-containing photoproducts with increasing GC content (from 28 to 72%) in isolated DNA. Thymine-thymine cyclobutane dimer was a minor lesion at high GC content. This trend was confirmed by an accurate and quantitative analysis of the photochemical data based on the exact dinucleotide frequencies of the studied genomes. The observation of the effect of the genome composition on the distribution of photoproducts was then confirmed in living cells, using two marine bacteria exhibiting different GC content. Because cytosine-containing photoproducts are highly mutagenic, it may be predicted that species with genomes exhibiting a high GC content are more susceptible to UV-induced mutagenesis.     

Cyclobutane pyrimidine dimers and (6-4) photoproducts block polymerization by DNA polymerase I

Bipyrimidine cyclobutane dimers and 6-4'-(pyrimidin-2'-one)-pyrimidine photoproducts are the major adducts formed in DNA following exposure to ultraviolet light. The relationship between the type and frequency of UV-induced DNA damage and the effects of such damage on DNA replication were investigated. UV-irradiated M13 phage DNA was employed in polymerization reactions with the Kenow fragment of Escherichia coli DNA polymerase I. The locations and frequencies of polymerase termination events occurring within a defined sequence of M13 DNA were compared with measurements of the locations and frequencies of UV-induced DNA damage of the same DNA sequence by using UV-specific enzymatic and chemical methods. The results indicate that both cyclobutane dimers and (6-4) photoproducts quantitatively block polymerization by DNA polymerase I.     

DNA lesion and mutagenesis induced in phageM13mp2 by UVA, UVB and UVC irradiation.

Sunlight is carcinogenic and mutagenic and its genotoxic effects are believed to be the result of UV light-induced lesions in DNA. These lesions include pyrimidine dimers and (6-4) photoproducts, but it is uncertain whether the pyrimidine modifications are the sole pre-mutagenic lesions induced by UV light. Previous studies indicate that some sunlight-induced mutations in the single-stranded DNA phage M13mp2 may not be caused by these photoproducts. In this work, purified single-stranded phage DNA was exposed to UVA, UVB and UVC and the induced mutations were analyzed. All 3 types of UV light increase the mutation frequency. The mutants were sequenced and the results suggest that UVA exposure may induce formation of a non-dipyrimidine lesion in DNA.     

Epidemiological study of sun exposure and visual field damage in children in Primorsko-Goranska County--the risk factors of earlier development of macular degeneration

The aim of this study was to examine the possible harmful effects of solar exposure on visual field damage in children living in Primorsko-Goranska County. Our previous work has shown noxious influence on visual field in children with anamnesis long term exposure to sunlight. This is an extended study, including children in Novi Vinodolski and Gorski kotar. We measured possible defect in isopteric visual field and macular-meridian thresholds. In the area of island of Rab these changes were the biggest, subsequently is Novi Vinodolski and at least Gorski kotar with the smallest range of eye complicates according to exposure to sunlight. These damages correlate with the areas of great solar emission. Damages in periphery isopters of visual field have shown the characteristics of periphery defect invagination, while increased macular thresholds in complete visual field was from 5 to 15Asb. We can conclude that there is direct connection between increased sunlight and long-term exposure to sunlight on one side, and on the other side, damages of retinal perception. Increased sun exposure may represent very important factors in early occurrence and develop of Age-Related Macular Degeneration (AMD). It is recommended the children protection in summer months, as well as taking derivates of vitamin A and antioxidants. Nowadays, AMD is one of the most important causes of damaged visual field, pretend to be national problem if we don't use the adequate prevention. World Health Organization has to begin with prevention of AMD, including these risk factors.     

Temporally distinct translesion synthesis pathways for ultraviolet light-induced photoproducts in the mammalian genome

Replicative polymerases (Pols) arrest at damaged DNA nucleotides, which induces ubiquitination of the DNA sliding clamp PCNA (PCNA-Ub) and DNA damage signaling. PCNA-Ub is associated with the recruitment or activation of translesion synthesis (TLS) DNA polymerases of the Y family that can bypass the lesions, thereby rescuing replication and preventing replication fork collapse and consequent formation of double-strand DNA breaks. Here, we have used gene-targeted mouse embryonic fibroblasts to perform a comprehensive study of the in vivo roles of PCNA-Ub and of the Y family TLS Pols η, ι, κ, Rev1 and the B family TLS Polζ in TLS and in the suppression of DNA damage signaling and genome instability after exposure to UV light. Our data indicate that TLS Pols ι and κ and the N-terminal BRCT domain of Rev1, that previously was implicated in the regulation of TLS, play minor roles in TLS of DNA photoproducts. PCNA-Ub is critical for an early TLS pathway that replicates both strongly helix-distorting (6-4) pyrimidine-pyrimidone ((6-4)PP) and mildly distorting cyclobutane pyrimidine dimer (CPD) photoproducts. The role of Polη is mainly restricted to early TLS of CPD photoproducts, whereas Rev1 and, in particular, Polζ are essential for the bypass of (6-4)PP photoproducts, both early and late after exposure. Thus, structurally distinct photoproducts at the mammalian genome are bypassed by different TLS Pols in temporally different, PCNA-Ub-dependent and independent fashions.