Disruption and functional analysis of six ORFs on chromosome IV: YDR013w, YDR014w, YDR015c, YDR018c, YDR020c, YDR021w (FAL1).

The disruption of six novel yeast genes has been realized in two genetic backgrounds. Six open reading frames (ORFs) from chromosome IV, YDR013w, YDR014w, YDR015c, YDR018c, YDR020c and YDR021w, were disrupted using the KanMX4 marker and PCR-targeting with long flanking regions homologous (LFH) to the target locus. The deletants were verified at the molecular level, using PCR and Southern analysis. Sporulation and tetrad analysis revealed that ORFs YDR013w and YDR021w (also known as FAL1) are essential genes. Microscopical observations showed that ydr013wDelta haploid cells were blocked after one or two cell cycles and presented heterogeneous bud sizes. The ydr021wDelta haploid cells gave rise to microcolonies of about 20 cells. The other four ORFs are non-essential. Basic phenotypic analysis of the non-lethal deletant strains did not reveal any significant differences in cell morphology, growth on different media and temperatures, sporulation and mating efficiency between parental and mutant strains in the FY1679 background.

Preferential incision of interstrand crosslinks induced by 8-methoxypsoralen plus UVA in yeast during the cell cycle.

Interstrand crosslink (ICL) induction by 8-methoxypsoralen plus UVA and the incision step of the repair have been investigated during the mitotic cell cycle of haploid Saccharomyces cerevisiae. Cells were synchronised by elutriation and events were examined at the level of the MAT alpha and the HML alpha loci in a SIR strain. The DNA sequence of these two loci is identical, but the MAT alpha locus may be replicated earlier in S phase and is transcriptionally active while the HML alpha locus may be replicated later in S phase and is transcriptionally inactive because of Sir repression that creates a heterochromatin-like structure at this locus. ICL were induced to similar extents in both loci during the stages of the cell cycle examined, and these levels were identical to those reported for asynchronous cultures. Preferential incisions occurred for ICL in the MAT alpha locus compared to those in the HML alpha locus, independently of the cell cycle phase studied. The levels of incision were comparable for events in the early G1 phase (eG1), late G1 phase (lG1), early S phase (eS), middle S phase (mS), late S phase (lS) or G2 phase (G2). Thus the preferential incision of ICL observed previously in asynchronous cell culture is maintained throughout the cell cycle and, surprisingly, occurs equally well in G1. Here the opportunities for recombination to further process the incised damaged are substantially limited compared to those in the S and G2 phases.

Preferential repair in Saccharomyces cerevisiae rad mutants after induction of interstrand cross-links by 8-methoxypsoralen plus UVA.

The gene specific induction and the incision step of the removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was measured in repair mutants of Saccharomyces cerevisiae. Events were examined at the MAT alpha and HML alpha loci in mutants deficient in the repair of ICL, namely rad1, rad2 delta, rad52, pso2 and the rad16 mutant which is impaired in the removal of UV-induced pyrimidine dimers from the silent HML alpha locus. Previously, we observed in a wild-type strain (K107) preferential repair concerning the incision of 8-MOP photo-induced ICL. The present study indicates that the two mutants rad1 and rad2 delta show no repair in either locus, due presumably to their deficiency in the incision step of ICL repair. The rad52 mutant which is defective in recombination, is proficient in the preferential incision of ICL at the MAT alpha locus versus the HML alpha locus. The same is true for the pso2 mutant which also lacks the ability to perform complete repair of ICL. The rad16 mutant is unable to repair ICL in the silent locus HML alpha but is proficient in repair (i.e. the incision of ICL) in the transcriptionally active MAT alpha locus.

Preferential repair in yeast after induction of interstrand cross-links by 8-methoxypsoralen plus UVA.

The gene-specific induction and removal of 8-methoxypsoralen (8-MOP) plus UVA-induced interstrand cross-links (ICL) was studied using the genetic system MAT alpha and HML alpha in Saccharomyces cerevisiae. We first examined events in a SIR alpha haploid strain (K107) in which these identical sequences are respectively transcriptionally active (MAT alpha) and inactive (HML alpha). Induction and repair of ICL was then studied in a sir3 mutant in which HML alpha is derepressed so that MAT alpha and HML alpha are both transcriptionally active. In the SIR strain at low levels of damage, no preferential repair of ICL occurred for MAT alpha versus HML alpha, whereas at high levels of ICL, those at MAT alpha were clearly repaired more rapidly than those at HML alpha. Similar experiments with the sir3 mutant revealed that the repair of ICL from both MAT alpha and HML alpha loci proceeded at the same rate at both low and high levels of damage. These data suggest that 8-MOP plus UVA-induced ICL are subject to preferential repair in yeast and that for the MAT alpha and HML alpha loci, this is dependent on their transcriptional status (i.e., the transcribed sequences are repaired more rapidly than the identical non-transcribed ones).

8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA.

Involvement of the PS03 gene of Saccharomyces cerevisiae in intrachromosomal mitotic recombination and gene amplification.

Using a genetic system of haploid strains of Saccharomyces cerevisiae carrying a duplication of the his4 region on chromosome III, the pso3-1 mutation was shown to decrease the rate of spontaneous mitotic intrachromosomal recombination 2- to 13-fold. As previously found for the rad52-1 mutant, the pso3-1 mutant is specifically affected in mitotic gene conversion. Moreover, both mutations reduce the frequency of spontaneous recombination. However, the two mutations differ in the extent to which they affect recombinations between either proximally or distally located markers on the two his4 heteroalleles. In addition, amplifications of the his4 region were detected in the pso3-1 mutant. We suggest that the appearance of these amplifications is a consequence of the inability of the pso3-1 mutant to perform mitotic gene conversion.

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.

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.

Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells.

In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e. monoadducts and interstrand crosslinks. Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences. We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest. In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA. The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido[3,4-c]psoralen greater than 3-carbethoxypsoralen. The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters. It probably involves an inducible component. Chromosome breakage occurs during the repair period after PUVA treatment. It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants.

The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was subcloned into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRNA sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.

Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae.

The RAD18 gene of Saccharomyces cerevisiae is involved in the error-prone DNA repair. Its nucleotide sequence, as reported here, predicts an open reading frame of 1461 nt which corresponds to a protein of 487 amino acids, with an Mr of 55,237. This protein has three putative zinc fingers, two acidic regions and a nucleotide-binding domain, suggesting that it is a nucleic acid-binding protein with a possible regulatory role.

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.

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.

[Absence of interstrand cross-links in DNA treated wtih 3-carbethoxypsoralen and 365 nm irradiation]. / Absence de pontages inter-chaînes dan l'ADN traité par le 3-carbéthoxypsoralène et une irradiation à 365 nm.

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.

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.

Biochemical analysis of damage induced in yeast by formaldehyde. II. Induction of cross-links between DNA and protein.

Exposure of Saccharomyces cerevisiae cells to formaldehyde-induced cross-links between DNA and proteins. This damage was demonstrated by three different techniques. Ultraviolet irradiation also produced cross-links between DNA and proteins in yeast.

Biochemical analysis of damage induced in yeast by formaldehyde. I. Induction of single-strand breaks in DNA and their repair.

Analysis of sedimentation profiles in alkaline sucrose gradients showed that, through a metabolic process, formaldehyde (FA) produced single-strand breaks in DNA of exponential phase cells of haploid wild-type Saccharomyces cerevisiae. The production of this type of lesion was dose-dependent. Strains defective in excision-repair of pyrimidine dimers induced by ultraviolet (UV) irradiation showed a reduced capacity to undergo single-stand breaks after treatment with FA. This indicates that the repair pathways of damage induced by UV and FA share a common step. Post-treatment incubation of wild-type cells in growth medium indicate a lag in cell division during which a slow recovery of DNA with a normal size was observed.

Yeast protoplasts from stationary and starved cells: preparation, ultrastructure and vacuolar development.

The conversion of stationary and starved yeast cells into protoplasts is described. The method is rapid, simple and can be applied to a variety of stationary yeast cells. Preincubation of yeast cells in the presence of pronase was essential for effective conversion into protoplasts. Baker's yeast and seven defined yeast strains, including one "petite", were studied. All of them were efficiently transformed into protoplasts in 60 to 90 min, depending on the strain culture conditions and the age of the culture. Protoplasts may be obtained even from late-stationary cells which contain spores. Saccharomyces cerevisiae cells subjected to complete starvation conditions in water, could also be completely transformed into protoplasts, even after 48 h of starvation. Electron microscope examination of stationary protoplasts from three different yeast strains showed no evidence of a remaining cell-wall. S. cerevisiae stationary cells show a very developed vacuolar system, a number of "lipid granules" and a few altered mitochondria. Endomycopsis fibuligera and Candida tropicalis stationary protoplasts show a similar fine structure, but "lipid granules" were completely absent.