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E Moustacchi

Publications and source records attributed to E Moustacchi.

At least 109 records · Page 6Linked to original sources
PubMed1983

Fate of photo-induced 8-methoxypsoralen mono-adducts in yeast. Evidence for bypass of these lesions in the absence of excision repair.

A fraction of UVA-induced 8-methoxypsoralen (8-MOP) mono-adducts can be transformed by a second UVA (365 nm) irradiation procedure into lethal cross-links in Saccharomyces cerevisiae. To follow the fate of cross-linkable mono-adducts, cells were incubated in complete medium between the two UVA doses and survival was measured. The killing effect of the second UVA dose decreases rapidly in haploid wild-type as well as in strains blocked in mutagenic (RAD6+ type) or in recombinogenic (RAD52+ type) repair pathways. This is also true in the pso1-1 and pso2-1 strains selected for sensitivity to 8-MOP plus UVA treatment. In contrast, persistence of mono-adducts is observed in strains blocked in the excision-resynthesis repair pathway. In other words, cross-linkable mono-adducts are repaired by the excision process. The use of the cell-cycle conditional mutant strain (cdc14-1) permitted us to apply the second dose at a specific cell-cycle stage (post-G2 phase) after a 'priming' UVA treatment on stationary (G1) phase cells. Such experiments showed a bypass of mono-adducts in an excision-deficient context for at least one round of DNA replication.

Cell Cycle↗
PubMed1983

Likelihood of the new antitumoral drug 10-[gamma-diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo [2,3-g]isoquinoline (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand breaks in vivo and its nonmutagenicity in yeast.

BD-40, a pyridopyrroloisoquinoline analogue of ellipticines, has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae. These inhibitory effects take place only in growing cells and are enhanced in the presence of oxygen. Among the different repair-deficient mutants examined, a mutant defective in DNA strand break repair (rad52-1) was found to be the most sensitive to such a toxic effect. A triple mutant blocked in the excision (rad2), the mutagenic (rad6), and the recombinogenic (rad52) repair pathways demonstrated the same sensitivity as the single rad52 mutant. Nuclear reversion and forward mutations as well as mitochondrial "petite" mutation were not induced by BD-40. These results indicate that: (a) the lesions induced in vivo by BD-40 are likely to be DNA strand breaks; (b) such damage is repairable in the wild type and is not of the mutagenic type; and (c) the excision pathway is not involved in such a repair of BD-40-induced lesions, and the mutagenic pathway plays a minor role. Since DNA strand breaks were not detected in vitro whether exposure of DNA to BD-40 was achieved in the presence or the absence of microsomal S-9 mix, it is suggested that an oxygen-dependent enzymatic processing, not linked to the microsomal monooxygenase complex, is required for the development of the cytotoxic activity of BD-40.

Aerobiosis↗
PubMed1982

Proteolytic activities in yeast after UV irradiation I. Variation in proteinase levels in repair proficient Rad+ strains.

Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD+ yeast cells after a dose of 50 Jm-2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD+ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.

Aminopeptidases↗
PubMed1982

Proteolytic activities in yeast after UV irradiation. II. Variation in proteinase levels in mutants blocked in DNA-repair pathways.

When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains. It is obvious that the induction of protease B activity following UV-treatment in Saccharomyces cannot be equated to the induction of the recA protein in Escherichia coli. However the correlation found between the block in mutagenic repair and the lack of UV-induction of protease B activity leads to questions on the possible role of certain protease activities in mutagenic repair in eucaryotic cells.

DNA Repair↗
PubMed1982

The fate of 8-methoxypsoralen photoinduced crosslinks in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair-deficient strains.

In Saccharomyces cerevisiae, after 8-methoxypsoralen [8-(OMe)Ps] photoaddition, more crosslinks are induced per unit dose in mitochondrial DNA than in nuclear DNA. In wild-type cells treated in the exponential phase of growth, single- and double-strand breaks are produced during crosslink removal and then are rejoined upon postexposure incubation. The incision step is almost blocked in the rad 3-2 mutant, which is also defective in excision-repair of UV-induced (254 nm) pyrimidine dimers. The cutting of crosslinks from nuclear DNA is depressed in wild-type stationary-phase cells. This is correlated with a higher sensitivity of such cells to 8-(OMe)Ps photoinduced cell killing. The incision of crosslinks is dramatically reduced in mitochondrial DNA. The rejoining of single- and double-strand breaks is not only dependent on the product of the RAD51 gene (as shown by others) but also of the PSO2 gene. A correlation was found between the ability to recombine and strand rejoining. Therefore, as in bacteria, both the excision and the recombinational repair systems are involved in crosslink repair in yeast. However, double-strand breaks in yeast constitute repair intermediates which are not detected in Escherichia coli. The LD37 (dose necessary to induce a mean of one lethal hit per cell) corresponds to about 120 crosslinks per genome in exponential-phase cells of the wild type and to 1-2 crosslinks in the pso2-1 mutant.

Cross-Linking Reagents↗
PubMed1981

Mutagenesis induced by mono- and bi-functional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso).

The inactivation and the induction of forward and reverse mutations by a mono- and a bifunctional nitrogen mustard in 3 pso mutants of Saccharomyces cerevisiae, initially selected for their sensitivity to psoralen photo-addition, were compared with that of the wild-type. The pso1-1 mutant was very sensitive to both alkylating agents, and the mutagenicity was abolished. This correlates with the defect in the error-prone repair capacity for lesions induced by psoralen photo-addition and radiations already observed for this mutant. Therefore it appears that the PSO1+ gene product acts on a spectrum of DNA lesions. The pso2-1 mutant was highly sensitive to the lethal effect of the bifunctional nitrogen mustard and was only slightly sensitive to the monofunctional one. For both agents a reduction in induced mutagenesis was seen. The same was true for mono- and bifunctional psoralen derivatives. The pso2-1 mutant having the same sensitivity as the wild-type to UV and ionizing radiations, it is suggested that the PSO2+ gene product is predominantly necessary for the repair of cross-links irrespective of their molecular nature. In contrast with psoralen photo-induced inactivation the pso3-1 mutant had the same sensitivity as the wild-type to alkylating agents. However, a reduction in induced mutagenesis was seen in both cases. This response was modulated according to dose and type of mutation. Consequently, it appeared that the PSO3+ gene product acts specifically on psoralen photo-induced sub-lethal lesions and on a fraction of premutagenic lesions independently of their structure.

Drug Resistance↗
PubMed1981

Interactions between mutations for sensitivity to psoralen photoaddition (pso) and to radiation (rad) in Saccharomyces cerevisiae.

The mode of interaction in haploid Saccharomyces cerevisiae of two pso mutations with each other and with rad mutations affected in their excision-resynthesis (rad3), error-prone (rad6), and deoxyribonucleic acid double-strand break (rad52) repair pathways was determined for various double mutant combinations. Survival data for 8-methoxypsoralen photoaddition, 254-nm ultraviolet light and gamma rays are presented. For 8-methoxypsoralen photoaddition, which induces both deoxyribonucleic acid interstrand cross-links and monoadditions, the pso1 mutation is epistatic to the rad6, rad52, and pso2 mutations, whereas it is synergistic to rad3. The pso2 mutation, which is specifically sensitive to photoaddition of psoralens, is epistatic to rad3 and demonstrates a nonepistatic interaction with rad6 and rad52. rad3 and rad6, as well as rad 6 and rad52, show synergistic interactions with each other, whereas rad 3 is epistatic to rad52. Consequently, it is proposed that PSO1 and RAD3 genes govern steps in the independent pathways. The PSO1 activity leading to an intermediate which is repaired via the three incidence pathways controlled by RAD6, RAD52, and PSO2 genes. Since pso1 interacts synergistically with rad3 and rad52 and epistatically with rad6 after UV radiation, the PSO1 gene appears to belong to the RAD6 group. For gamma ray sensitivity, pso1 is epistatic to rad6 and rad52, which suggests that this gene controls a step which is common to the two other independent pathways.

Epistasis, Genetic↗
PubMed1980

[Absence of interstrand cross-links in DNA treated wtih 3-carbethoxypsoralen and 365 nm irradiation].

In contrast to 8-MOP photoaddition on DNA, the renaturation capacity of heat or alkali denatured DNA treated with 3-CPs is absent. This shows that DNA interstrand cross-links are not produced by 3-CPs photoaddition even by a dose which leads to 10(-5) survivors. At equimolar concentrations of 3-CPs or 8-MOP (5 x 10(-4) M) and a dose of 10 kJ.m-2 of 365 nm radiation, 1 molecule of 3-CPs for 4 x 10(3) nucleotides and 1 molecules of 8-MOP for 9 x 10(3) nucleotides are fixed in vivo. Since wild type cells are about 4 times more sensitive to 8-MOP than to 3-CPs photoaddition, it implies that cross-links are important in genotoxicity and that monoadditions are more easily repaired than cross-links.

DNA, Fungal↗
PubMed1980

Mutagenicity testing with eukaryotic microorganisms.

The different genetic end-points which can be tested to detect genotoxicity of chemicals in fungi and especially in the yeast Saccharomyces cerevisiae are described. They include reversion and forward mutation, mitochondrial deletions and point mutations, mitotic or meiotic intra- and intergenic recombination, chromosomal non-disjunction and aneuploidy. Several factors known to affect the response to genotoxic agents such as the growth parameters, the repair ability, the cells permeability, etc., are discussed. The recent validation studies on the mutagenic and recombinogenic activities of a number of chemicals indicate that within the battery of rapid, low cost and quantitatively reliable tests, the yeast system can be profitably used.

Bacteria↗
PubMed1980

Biochemical analysis of damage induced in yeast by formaldehyde.

Cross-links between DNA and proteins were induced by formaldehyde treatment in yeast cells. This damage can be repaired by post-treatment incubation of cells or protoplasts in nutrient medium. This repair was observed for wild-type cells as well as for a UV-sensitive, excision-deficient mutant (rad1-3), also sensitive to the lethal effect of formaldehyde.

DNA Repair↗
PubMed1980

Yeast mitochondrial DNA characterization after ultraviolet irradiation.

Yeast mitochondrial (mtDNA) 3H-labelled was isolated from exponential phase cells after ultraviolet light irradiation. Both the size and amount of mtDNA were found to be reduced during a 40-h liquid-holding (LH) period in non-growth medium following irradiation as compared to the mtDNA recovered from nonirradiated cells under similar conditions. After the LH period, previously irradiated cells were resuspended in growth medium containing [14C]adenine. Double labelled mtDNA (3H and 14C) was isolated from cell samples removed during new growth. A recovery in the amount and size of mtDNA was observed in irradiated cells during new growth. These biochemical studies agree with the observed loss and recovery of mtDNA genetic markers in UV-irradiated exponential phase yeast after a period of LH and new growth resp.

DNA Replication↗
PubMed1980

Isolation and characterization of pso mutants sensitive to photo-addition of psoralen derivatives in Saccharomyces cerevisiae.

We have isolated mutants sensitive to photo-addition of bi-functional and mono-functional derivatives of psoralen in Saccharomyces cerevisiae. Three of these pso mutants were analyzed in detail. They segregate in meiosis like Mendelian genes and complement each other, as well as existing radiation-sensitive (rad and rev) mutants. The study of heterozygous diploid strains (PSO+/pso) indicates that the three pso genes are recessive. The mutant pso1--1 demonstrates a cross-sensitivity to UV and gamma-rays, whereas mutants pso2--1 and pso3--1 are specifically sensitive to photo-addition of psoralen derivatives. The comparison of exponentially growing cells to stationary-phase cells demonstrates that for the three mutants the defect in repair capacity of DNA cross-links and monoadducts concerns G1 and early S-phase cells. The pso2--1 mutant is, however, also defective in G2 repair and loses diploid resistance when it is in the homozygous state.--The block in repair capacity in these novel mutants is discussed in relation to the three other repair pathways known to be involved in the repair of furocoumarins photo-induced lesions in yeast DNA.

Cell Cycle↗
PubMed1980

The effects of three PSO genes on induced mutagenesis : a novel class of mutationally defective yeast.

Reverse and forward mutation, induced by photoaddition of 8-methoxypsoralen (8-MOP) and 3-carbethoxypsoralen (3-CPs) or ultraviolet light (UV), are reduced in three pso mutants of Saccharomyces cerevisiae. The pso1-1 strain exhibits a lower frequency of spontaneous reversion (anti-mutator) and is almost entirely unaffected by the three agents in both the haploid and diploid states. The pso2-1 strain demonstrates very reduced frequencies of 8-MOP and 3-CPs plus 365 nm radiation-induced mutations in haploid and diploid cells. UV-induced mutation are slightly reduced, whereas survival is almost normal. The pso3-1 strain is mutable by 8-MOP and 3-CPs photoaddition only in the low-dose range. After UV treatment, survival of pso3-1 is nearly normal, whereas the frequencies of induced mutants are diminished as compared to the normal PSO+. An analogue of adenine, 6-N-hydroxyaminopurine, is capable of inducing reversions in wild type, as well as in pso and rad6-1 mutant strains, indicating that this drug may act as a direct mutagen in yeast. The comparison of photoaddition of the bifunctional agent (8-MOP) to that of the monofunctional one (3-CPs) confirms that cross-links, as well as monoadditions, are mutagenic in S. cerevisiae. Repair, of the recombinational type, taking place in diploid cells or in haploid cells in G2 phase leads to higher survival, but appears to be error-free.

DNA Repair↗
PubMed1979

Abolition of the cyclic variations in radiosensitivity during meiosis in a sporulation mutant blocked in premeiotic DNA synthesis.

The response to ultraviolet light (254 nm) of two sporulation mutants during the meiotic process was compared to that of a wild type diploid strain of Saccharomyces cerevisiae. The cyclic pattern for cell killing and rho- induction characteristic of diploid wild type cells persists in a strain able to perform the premeiotic DNA synthesis but which is blocked in the further steps of meiosis (spo8 DMS1). On the contrary, these fluctations are abolished in a derived mutant (spo8 dsm1) which is blocked in the premeiotic DNA synthesis. Under these conditions, the response to cell killing can be dissociated from the observed for rho- induction.

DNA↗
PubMed1979

Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation.

In normal human fibroblasts we observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradiation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C.

Cell Line↗
PubMed1979

Genetic effect of 3-carbethoxypsoralen, angelicin, psoralen and 8-methoxypsoralen plus 365-nm irradiation in Saccharomyces cerevisiae: induction of reversions, mitotic crossing-over, gene conversion and cytoplasmic "petite" mutations.

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 X 10(-5) M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m-2 min-1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photo-addition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic "petite" mutation in stationary phase cells per unit irradiation dose or per viable cell.

Alleles↗
PubMed1979

The fate of mitochondrial loci in rho minus mutants induced by ultraviolet irradiation of Saccharomyces cerevisiae: effects of different post-irradiation treatments.

Three main features regarding the loss of mitochondrial genetic markers among rho- mutants induced by ultraviolet irradiation are reported: (a) the frequency of loss of six loci examined increases with UV dose; (b) preferential loss of one region of the mitochondrial genome observed in spontaneous rho- mutants is enhanced by UV; and (c) the loss of each marker results from large deletions. Marker loss in rho- mutants was also investigated under conditions that modulate rho- induction. Liquid holding of irradiated exponential or stationary phase cells, as well as a split-dose regime applied to stationary phase cells, results in rho- mutants in which the loss of markers is correlated with rho- induction: the more sensitive the cells are to rho- induction, the more frequent are the marker losses among rho- clones derived from these cells. This correlation is not found in exponential-phase cells submitted to a split-dose treatment, suggesting that a different mechanism is involved in the latter case. It is known that UV-induced pyrimidine dimers are not excised in a controlled manner in mitochondrial DNA. However, our studies indicate that an accurate repair mechanism (of the recombinational type ?) can lead to the restoration of mitochondrial genetic information in growing cells.

Cell Division↗