- Email: [email protected]
Repair of monoadducts induced by photoaddition of furocoumarins in yeast. Comparison of monofunctional and bifunctional agents
200 1.5.11 Maenhaut-Michel, G., and P. Caillet-Fauquet, Drpartement de Biologie Molrculaire, Universit6 Libre de Bruxelles, Brussels (Belgium)
Genetic control of targeted and untargeted mutagenesis Mutagenesis induced by ultraviolet irradiation and by many chemicals depends upon transient induction of a cellular mutator effect (SOS mutator activity) which permits fixation of mutations opposite DNA lesions (targeted mutagenesis) as well as on undamaged DNA (untargeted mutagenesis). Two different mutation systems were used to measure the SOS mutator activity in E. coli; (i) forward mutagenesis in the immunity control region of bacteriophage ~, (c + ~ c) and (ii) base substitution mutagenesis resulting in reversion of an amber codon in the ~R gene. Targeted and untargeted mutagenesis were analyzed in different E. coli mutants which affect (i) excision repair (uvrA, uvrB), (ii) recombination repair (recA, recF), (iii) SOS induction (recA, lexA), (iv) methyl-directed post-replicative mismatch repair (mutH, mutL, routS, dam). Mutagenesis induced by UV irradiation of the host bacteria in unirradiated or weakly irradiated h phages (untargeted mutagenesis) as well as in heavily irradiated ~ phage (targeted mutagenesis) is dependent on recA and lexA genes. Untargeted mutagenesis is increased in mutH, mutL and routS mutants, decreased in dam, uvrA, uvrB and reeF mutants and not affected in umuC mutants. Targeted mutagenesis is decreased in recF and umuC mutants and is not affected in dam, mutH, mutL, routS and in uvrA or uvrB mutants. These results indicate that untargeted mutagenesis may be the consequence of a recA, lexA-dependent decreased fidelity of DNA replication which does not require the umuC function. Mutagenesis of DNA containing a high level of lesions (targeted mutagenesis) requires the umuC gene product and is not affected by the mismatch correction system.
1.5.12 Magana-Schwencke, N., and E. Moustacchi, Institut Curie-Biologie, Centre Universitaire, Bat. 110, 91405 Orsay (France)
Repair of monoadducts induced by photoaddition of furocoumarins in yeast. Comparison of monofunctional and bifunctional agents Monoadducts induced by photoaddition of furocoumarins are less mutagenic and recombinogenic than interstrand crosslinks in yeast. The use of monofunctional compounds may be of interest in therapy to prevent genetic hazards. Nevertheless, the molecular steps of the repair of monoadducts are not yet well established. We present data obtained with two monofunctional agents: 3-carbethoxypsoralen (3-CPs) and 7-methylpyridopsoralen (lb), and a bifunctional agent: 8-methoxypsoralen (8-MOP). The fixation on yeast DNA of lb is 2 times greater than 3-CPs and almost 5 times greater than 8-MOP. However, the phototoxicity obtained with lb is greater than that obtained with 3-CPs, but is similar to that obtained with 8-MOP. The induction and repair of single-strand breaks during post-treatment incubation of cells in complete medium was measured. In wild type after 3-CPs treatment, the appearance of single-strand breaks is difficult to measure because their repair is rapid. After 8-MOP treatment, single-strand breaks are detected 15 rain after treatment and they are repaired within 2 h of post-treatment incubation. When using lb, the single-strand breaks detected are not repaired within 4 h of post-treatment incubation. The disappearance of labelled lb, 3-CPs and 8-MOP during this incubation from DNA was followed, in wild-type strain and
201
mutants defective in repair. The fate of lesions appears to differ according to the nature of the monofunctional agent.
1.5.13 Moustacchi, E., V. Favaudon and E. Bisagni, Institut Curie-Biologie, Centre Universitaire, BM. 110, 91405 Orsay (France) A rapid cytotoxic assay on wild-type and specific repair-deficient yeast allows prediction on the nature of lesions produced by new drugs A rapid cytotoxicity and repair assay which allows to predict the in vivo mode of action of new drugs has been developed in Saccharomyces cerevisiae. If a mutant blocked in the repair of a specific type of lesion is more sensitive than the wild type, it can be inferred that the agent tested is likely to induce principally the specific unrepairable lesion. This test then guides the biochemical detection of the predicted lesion(s). Differential sensitivity of RAD + and rad- (or pso-) is measured by comparing growth in serial concentration of the tested drug for a constant period of incubation. Using this assay, we show that neocarcinostatin induces in vivo repairable DNA strand breaks in wild-type cells [1]. The same is true for a new ellipticine derivative, BD40, a promising antitumoral drug in view of its non-mutagenicity in yeast and Salmonella [2]. The response of the pso2 mutant in comparison to the isogenic wild type to 8 known cross-linking or monofunctional derivatives (nitrogen mustards, mitomycin, psoralens, etc.) was studied. The pso2 mutant turned out to be sensitive only to the bifunctional agents and biochemically it demonstrated a specific defect in cross-link repair. On the basis of this validation of the test, the sensitivity of pso2 to 5 new pyridopsoralens plus UVA was studied and the functionality in vivo of the new compounds was consequently established. Two particularly interesting compounds in view of their photochemotherapeutic properties (Dubertret, personal communication), the pyrido [3,4-c]furo[3,2g]coumarin and its 7-methyl derivative were found to be monofunctional. References 1 Moustacchi, E., and V. Favaudon, Mutation Res., 104 (1982) 87-94. 2 Moustacchi, E., V. Favaudon and E. Bisagni, Cancer Res., 43 (1983) 3700-3706.
1.5.14 Poirier, V., J.F. Burke and M.R. James, MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BN1 9QG (Great Britain) Expression of a yeast DNA ligase gene in mammalian cells A cell line, termed 46BR, derived from an immunodeficient individual is unusually sensitive to a number of alkylating agents. A biochemical study has been undertaken and it appears that the DNA ligation step is defective in this cell line. We are attempting to complement this defect in 46BR using a yeast gene introduced into these cells. We are interested in the transient expression of the yeast ligase gene using its own promoter or the SV40 promoter and the continued expression of the gene.
Genetic control of targeted and untargeted mutagenesis Mutagenesis induced by ultraviolet irradiation and by many chemicals depends upon transient induction of a cellular mutator effect (SOS mutator activity) which permits fixation of mutations opposite DNA lesions (targeted mutagenesis) as well as on undamaged DNA (untargeted mutagenesis). Two different mutation systems were used to measure the SOS mutator activity in E. coli; (i) forward mutagenesis in the immunity control region of bacteriophage ~, (c + ~ c) and (ii) base substitution mutagenesis resulting in reversion of an amber codon in the ~R gene. Targeted and untargeted mutagenesis were analyzed in different E. coli mutants which affect (i) excision repair (uvrA, uvrB), (ii) recombination repair (recA, recF), (iii) SOS induction (recA, lexA), (iv) methyl-directed post-replicative mismatch repair (mutH, mutL, routS, dam). Mutagenesis induced by UV irradiation of the host bacteria in unirradiated or weakly irradiated h phages (untargeted mutagenesis) as well as in heavily irradiated ~ phage (targeted mutagenesis) is dependent on recA and lexA genes. Untargeted mutagenesis is increased in mutH, mutL and routS mutants, decreased in dam, uvrA, uvrB and reeF mutants and not affected in umuC mutants. Targeted mutagenesis is decreased in recF and umuC mutants and is not affected in dam, mutH, mutL, routS and in uvrA or uvrB mutants. These results indicate that untargeted mutagenesis may be the consequence of a recA, lexA-dependent decreased fidelity of DNA replication which does not require the umuC function. Mutagenesis of DNA containing a high level of lesions (targeted mutagenesis) requires the umuC gene product and is not affected by the mismatch correction system.
1.5.12 Magana-Schwencke, N., and E. Moustacchi, Institut Curie-Biologie, Centre Universitaire, Bat. 110, 91405 Orsay (France)
Repair of monoadducts induced by photoaddition of furocoumarins in yeast. Comparison of monofunctional and bifunctional agents Monoadducts induced by photoaddition of furocoumarins are less mutagenic and recombinogenic than interstrand crosslinks in yeast. The use of monofunctional compounds may be of interest in therapy to prevent genetic hazards. Nevertheless, the molecular steps of the repair of monoadducts are not yet well established. We present data obtained with two monofunctional agents: 3-carbethoxypsoralen (3-CPs) and 7-methylpyridopsoralen (lb), and a bifunctional agent: 8-methoxypsoralen (8-MOP). The fixation on yeast DNA of lb is 2 times greater than 3-CPs and almost 5 times greater than 8-MOP. However, the phototoxicity obtained with lb is greater than that obtained with 3-CPs, but is similar to that obtained with 8-MOP. The induction and repair of single-strand breaks during post-treatment incubation of cells in complete medium was measured. In wild type after 3-CPs treatment, the appearance of single-strand breaks is difficult to measure because their repair is rapid. After 8-MOP treatment, single-strand breaks are detected 15 rain after treatment and they are repaired within 2 h of post-treatment incubation. When using lb, the single-strand breaks detected are not repaired within 4 h of post-treatment incubation. The disappearance of labelled lb, 3-CPs and 8-MOP during this incubation from DNA was followed, in wild-type strain and
201
mutants defective in repair. The fate of lesions appears to differ according to the nature of the monofunctional agent.
1.5.13 Moustacchi, E., V. Favaudon and E. Bisagni, Institut Curie-Biologie, Centre Universitaire, BM. 110, 91405 Orsay (France) A rapid cytotoxic assay on wild-type and specific repair-deficient yeast allows prediction on the nature of lesions produced by new drugs A rapid cytotoxicity and repair assay which allows to predict the in vivo mode of action of new drugs has been developed in Saccharomyces cerevisiae. If a mutant blocked in the repair of a specific type of lesion is more sensitive than the wild type, it can be inferred that the agent tested is likely to induce principally the specific unrepairable lesion. This test then guides the biochemical detection of the predicted lesion(s). Differential sensitivity of RAD + and rad- (or pso-) is measured by comparing growth in serial concentration of the tested drug for a constant period of incubation. Using this assay, we show that neocarcinostatin induces in vivo repairable DNA strand breaks in wild-type cells [1]. The same is true for a new ellipticine derivative, BD40, a promising antitumoral drug in view of its non-mutagenicity in yeast and Salmonella [2]. The response of the pso2 mutant in comparison to the isogenic wild type to 8 known cross-linking or monofunctional derivatives (nitrogen mustards, mitomycin, psoralens, etc.) was studied. The pso2 mutant turned out to be sensitive only to the bifunctional agents and biochemically it demonstrated a specific defect in cross-link repair. On the basis of this validation of the test, the sensitivity of pso2 to 5 new pyridopsoralens plus UVA was studied and the functionality in vivo of the new compounds was consequently established. Two particularly interesting compounds in view of their photochemotherapeutic properties (Dubertret, personal communication), the pyrido [3,4-c]furo[3,2g]coumarin and its 7-methyl derivative were found to be monofunctional. References 1 Moustacchi, E., and V. Favaudon, Mutation Res., 104 (1982) 87-94. 2 Moustacchi, E., V. Favaudon and E. Bisagni, Cancer Res., 43 (1983) 3700-3706.
1.5.14 Poirier, V., J.F. Burke and M.R. James, MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton BN1 9QG (Great Britain) Expression of a yeast DNA ligase gene in mammalian cells A cell line, termed 46BR, derived from an immunodeficient individual is unusually sensitive to a number of alkylating agents. A biochemical study has been undertaken and it appears that the DNA ligation step is defective in this cell line. We are attempting to complement this defect in 46BR using a yeast gene introduced into these cells. We are interested in the transient expression of the yeast ligase gene using its own promoter or the SV40 promoter and the continued expression of the gene.