Alain Gentil

RETRACTED: Transcription-Coupled Repair of 8-oxoGuanine

Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.

Apurinic sites cause mutations in simian virus 40.

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.

Mutation spectra induced by replication of two vicinal oxidative DNA lesions in mammalian cells.

Ionizing radiations often induce multiple and clustered DNA lesions at the site of DNA interaction. As a model, we have studied the toxicity and the mutagenicity of two adjacent oxidative bases as clustered DNA lesions in mammalian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo-7,8-dihydroguanine, the formylamine residue resulting from the oxidation of a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylamine where both modifications are located at a vicinal position. A single-stranded DNA shuttle vector carrying a unique DNA lesion was constructed, transfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as expected, does not affect greatly survival (70% bypass) whereas formylamine and the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studied. Under our experimental conditions, replication of 8-oxo-7, 8-dihydroguanine finally gives rise to guanine:cytosine pairing, rendering this lesion only slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were observed targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding properties of the formylamine render this residue very mutagenic when coming from the oxidative alteration of a cytosine. The coding properties of the tandem damage are a combination of the contribution of the two isolated lesions with a very high percentage of adenine insertion (94%) opposite the formylamine residue of the tandem lesion. The toxicity as well as the mutation spectrum of the tandem lesion allow us to speculate about the molecular mechanism with which the DNA polymerase replicates these two lesions.

Repair and mutagenesis survey of 8-hydroxyguanine in bacteria and human cells

8-Hydroxyguanine is one of the major products formed by the reactive oxygen species which are generated in living cells as a consequence of either the normal metabolic pathways or an exogeneous chemical or physical stress. The production of the oxidative damage is described and the different repair pathways of the oxidative lesions are analyzed from bacteria to human cells. Analysis of repair in human cells harboring different deficiencies in the nucleotide excision repair mechanism such as xeroderma pigmentosum cells from different complementation groups and cells from Cockaynes syndrome patients allows us to emphasize the possibility of the intervention of this repair mechanism on the elimination of oxidative damages. Finally, a repair model of oxidative lesions is proposed.

Enhanced reactivation and mutagenesis after transfection of carcinogen-treated monkey kidney cells with UV-irradiated Simian Virus 40 (SV40) DNA

Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.

Implication of uracil in spontaneous mutagenesis on a single-stranded shuttle vector replicated in mammalian cells

Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.

Strategies to Analyse Mutagenesis in Mammalian Cells Using Simian Virus 40 or Shuttle Vectors

SUMMARY The use of exogeneous DNA probes, which replicate extrachromosomally, is proposed in order to study spontaneous and induced mutagenesis in mammalian cells. Simian virus 40 has already proved to be very useful, since it has provided much important information in this field. Recently, several shuttle vectors have been designed for this purpose; however, it seems that these molecules have high spontaneous mutation frequencies when replicating in mammalian cells. We have developed new alternative systems, such as Epstein-Barr virus-based shuttle vectors that can be episomally maintained in human cells. Furthermore, we have constructed packageable shuttle vectors, which appear to be stable in the host cell and thus suitable for analysis of mutagenesis.

Mechanisms and Consequences of Mutation Induction in Mammalian Cells

Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species. Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells. However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years. Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells. These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells. In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage. This has been well characterized using ultraviolet (UV) light as a mutagen. UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced. The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours. Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations. A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin. This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum. The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions.

Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts.

Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.