Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions. We present herein further results obtained in order to explore that possibility, together with a more general report on the subject. pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E. coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined. Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway. A mutagenic repair pathway did not show a significant contribution to the total repair process. Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred. Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA. In contrast, the recA dependent pathway was needed only for SOS induction. After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved.     

Repair of cis-platinum-DNA adducts by ABC excinuclease in vivo and in vitro.

cis-Platinum compounds, which are used in cancer chemotherapy, are thought to exert their effect by damaging DNA. It is known that this damage is partially repaired in Escherichia coli. Using cis-Pt-treated pBR322 DNA as a probe, we investigated the role of nucleotide excision repair in the removal of Pt-DNA adducts. We found that the nucleotide excision pathway was the major mechanism for repairing Pt adducts in transforming plasmid DNA but that a recA-dependent pathway also contributed to plasmid survival. When cis-Pt-damaged pBR322 was treated with the purified nucleotide excision enzyme ABC excinuclease in vitro, a fraction of the adducts was removed by the enzyme; this removal resulted in a corresponding increase in transformation efficiency.     

Repair of plasmid DNA treated with 8-methoxypsoralen and long-wave UV light (lambda=365 nm) in wild type and mutant rad2 cells of Saccharomyces cerevisiae

The method of repeated irradiation has been used to study excision of 8-MOP monoadducts from plasmid and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurement of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excised in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmid treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 degrees C, prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmid DNA, but this process is blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA.     

W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

Mutation induction and killing of Escherichia coli by DNA adducts and crosslinks: a photobiological study with 8-methoxypsoralen.

Low doses of 350 nm radiation (NUV) in the presence of 8-methoxypsoralen (8-MOP) induce predominantly mono-adducts in bacterial DNA. Further exposure to NUV in the absence of 8-MOP converts a proportion of these mono-adducts to interstrand cross-links. Using this approach the relative effects of adducts and cross-links on bacteria with different repair capacities was studied. Escherichia coli WP100 uvrA recA, believed to be totally deficient in the ability to repair 8-MOP plus NUV damage to DNA, was inactivated on average by a single photon event occurring with a quantum efficiency of about 0.03. We conclude that the inactivating lesion is probably a single mono-adduct. E. coli WP2 uvrA, deficient in excision endonuclease activity, may be inactivated by a very small number of cross-links, probably one. These conclusions are consistent with present knowledge of the repair capabilities of these bacteria. Conversion of mono-adducts to cross-links in WP2 uvrA (which occurs with a quantum efficiency of around 0.3) greatly increases lethality but results in a reduction of the induced mutation frequency presumably because cross-links are (almost) invariably lethal. In the repair-proficient strain WP2 both adducts and cross-links can be repaired but the latter are more likely than the former to lead to either death or mutation.     

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.     

Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.     

Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxic effects in diploid yeast

The induction of genetic effects was studied in a diploid strain of Saccharomyces cerevisiae (D7) after treatments with the monofunctional furocoumarins 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[3,4-c]psoralen (PyPs) and 3-carbethoxypsoralen (3-CPs) and the bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of 365-nm radiation. The DNA photobinding of radioactively labelled MePyPs, 3-CPs, 5-MOP and 8-MOP was determined in parallel. The DNA-photobinding capacity was highest for MePyPs followed in decreasing order by 5-MOP, 3-CPs and 8-MOP. At a concentration of 5 microM and 4.2 kJ/m2 of 365-nm radiation approximately 160, 66, 60 and 16 adducts per 10(6) base pairs were formed by MePyPs, 5-MOP, 3-CPs and 8-MOP, respectively. The activity of MePyPs and PyPs for the induction of lethal effects lay in the same range as that of 5-MOP whereas 8-MOP was 3 times less active and 3-CPs showed very little activity. For the induction of mitotic gene conversion and genetically altered colonies including mitotic crossing-over the order of activity was about the same as that observed for the induction of lethal effects: MePyPs greater than 5-MOP greater than PyPs greater than 8-MOP much greater than 3-CPs. Nuclear reversions were induced most effectively by 5-MOP, 8-MOP being about 3 times less effective. Up to 4 and 6 kJ/m2 of 365-nm radiation, MePyPs and PyPs, respectively, were less mutagenic than 8-MOP but became more mutagenic at higher doses. At equal survival, the pyridopsoralens were, however, clearly less mutagenic than the bifunctional furocoumarins 8-MOP and 5-MOP. By plotting the genetic data versus the number of lesions induced in DNA, it was shown that the monoadducts induced by the monofunctional furocoumarins MePyPs and 3-CPs exert a relatively low potential for the induction of lethal and nuclear genetic events as compared to photoadditions induced by the bifunctional furocoumarins 8-MOP and 5-MOP. However, at a very high density, the monoadducts induced by MePyPs became as lethal and as mutagenic as the mixture of mono- and biadducts induced by 8-MOP and 5-MOP probably due to overloading of cellular repair capacities.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA photobinding of 7-methylpyrido[3,4-c]psoralen and 8-methoxypsoralen. Effects on macromolecular synthesis, repair and survival in cultured human cells

The photobinding to DNA of tritiated 7-methylpyrido[3,4-c]psoralen (MPP), a recently synthesized monofunctional compound of therapeutical interest, and of 8-methoxypsoralen (8-MOP) was determined in cultured normal human fibroblasts. Employing compounds at 10(-6) M, MPP photobinds approximately 11 times more efficiently than 8-MOP: one molecule is fixed respectively per 7.5 X 10(4) or 8.1 X 10(5) base pairs/kJ . m-2 of 365-nm radiation (UVA). Removal of bound material from DNA is slow and limited in 48-72 h of post-treatment incubation to 30-40% of initial adducts formed by MPP and to 50-60% of those of 8-MOP. For equivalent photobinding MPP and 8-MOP induce similar inhibitions of DNA synthesis. However, the recovery of DNA synthesis during post-treatment incubation is lower after photoaddition of MPP than after that of 8-MOP. MPP also exerts a much higher lethal effect than 8-MOP: one lethal hit corresponds to about 4400 and to 19,900 adducts per cell respectively. Alkaline elution experiments confirmed the monofunctional nature of MPP and indicated that in MPP-damaged cells DNA breaks accumulate with time of post-treatment incubation. In contrast, after photoaddition of 3-carbethoxypsoralen (3-CPs), another monofunctional furocoumarin, or irradiation with 254-nm UV, DNA breaks are induced only transiently. In 8-MOP-treated cells, DNA cross-links appear to be partially repaired. In conclusion, MPP monoadducts turn out to constitute more cytotoxic lesions than 8-MOP mono- and bi-adducts.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.     

Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerevisiae

The contribution of different repair pathways to the repair of 8-methoxypsoralen (8-MOP) plus UVA induced lesions on a centromeric plasmid (YCp50) was investigated in the yeast Saccharomyces cerevisiae using the lithium acetate transformation method. The pathways of excision-resynthesis (RAD1) and recombination (RAD52) were found to be involved in the repair of exogenous as well as of genomic DNA. Mutants in RAD6 and PSO2 genes showed the same transformation efficiency with 8-MOP plus UVA treated plasmid as wild-type cells suggesting that these latter pathways involved in mutagenesis are not operating on plasmid DNA although required for the repair of 8-MOP photoadducts induced in genomic DNA. These results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the repair system and the nature of the DNA damage considered.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.     

Real-time fluorescence-based detection of furanocoumarin photoadducts of DNA

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (lambda = 320-400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts.     

Sensitized NADH formation of single-stranded breaks in plasmid DNA upon the action of near UV-radiation

It has been shown that NADH photosensitize in vitro single-strand breaks formation in double-strand plasmid DNA pBR 322 upon near-UV (320-400 nm) irradiation. The number of single-strand breaks depends both on UV light dose and sensitizer concentration. Addition of catalase and sodium benzoate strongly decreases the single-strand breaks formation. The results show an important role of hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in inducing single-strand breaks in plasmid DNA irradiated by near-UV radiation in the presence of NADH.     

Further characterization of an E. coli strain resistant to the toxic and mutagenic action of cis-diamminedichloroplatinum(II)

An increased resistance to the toxic and mutagenic activity of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) in the E. coli strain BS21 compared to its wild-type parent, F26, has been reported. This resistance was neither due to different binding of cis-DDP to DNA nor to adaptive DNA repair (Germanier et al., 1984) In the present work, we found that mutation of the uvrA, recA and polA genes did not abolish the resistance of BS21 to the toxic action of cis-DDP. The lower mutability of BS21 was not influenced by the polA mutation, while uvrA greatly reduced and recA eliminated the mutagenic activity of cis-DDP in both strains. Treatment of BS21 and F26 with equal doses of cis-DDP produced the same initial number of platinum-DNA lesions. Little excision repair was detected in vivo in either strain during 6-h post-treatment incubation, the F26 strain being the most efficient of the two for this process. In contrast, F26 and BS21 were transformed identically by pBR322 DNA which had been treated with cis-DDP in vitro. Analysis of the platinum-DNA adducts which were formed between cis-DDP and salmon sperm DNA in the buffer conditions of this experiment suggests that plasmid DNA contains 80% monofunctional adducts and 20% bifunctional bis-guanine adducts.These data indicate that the selective toxicity and mutagenicity of these two strains in vivo are neither a result of different numbers of Pt-DNA lesions nor of their repair. The selectivity disappeared when the two bacterial strains were transformed by pBR322 DNA containing identical platinum-DNA lesions, suggesting that the biochemical events which process platinum-DNA lesions are the same in both strains. Hence, it appears that cis-DDP may form qualitatively different platinum-DNA adducts in the BS21 and F26 strains which are responsible for the different toxicity and mutagenicity.     

Genetic control of the bypass of mono-adducts and of the repair of cross-links photoinduced by 8-methoxypsoralen in yeast

A large UVA dose by itself induces lethal damage revealed in some repair-deficient strains of Saccharomyces cerevisiae. Following photoaddition of a monofunctional psoralen derivative, 3-carbethoxypsoralen, an extra killing effect is observed by applying a second high UVA dose, in conditions where a fraction of 8-methoxypsoralen (8-MOP) plus UVA-induced monoadducts are transformed into DNA cross-links. In an excision-repair-deficient context, the bypass of 8-MOP plus UVA-induced monoadducts is under the control of the RAD6+ gene product. However, when other steps of the mutagenic pathway are blocked by the rad18-2 or the pso1-1 mutations, bypass occurs. This is also true when in excision-deficient strains the recombinogenic pathway is blocked by the rad52-1 mutation. The recombinogenic pathway may be an alternative to the mutagenic pathway for bypass of monoadducts. The repair of the lesions induced by a second UVA dose applied after a first treatment by 8-MOP plus UVA [i.e. cross-links and other putative lesion(s)] is controlled by at least the RAD2+, RAD6+, RAD52+, PSO2+ and PSO1+ gene products. The role of the pathways involved is discussed according to the nature of the secondarily induced lesions.