In, Saccharomyce8 cerevisiae, after 8-methoxypsoralen 18-(OMe)Ps] photoaddition, more crosslinks are induced per unit dose in mitochondrial DNA than in nuclear DNA. In wildtype 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 rad3-2 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 ofsingle-and double-strand breaks is not only dependent on the product of the RAD51 gene (as shown by others) but also of the PS02 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 Eacherichia 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 p8o2-1 mutant.The psoralen derivative, 8-methoxypsoralen [8-(OMe)Ps] intercalates in DNA; upon exposure to near UV radiation, it reacts with pyrimidine bases to form monoadducts and interstrand crosslinks (1, 2). These photomodifications of DNA lead to lethality and induction of mutation and recombination (1-3). In addition, the production in yeast of the mitochondrial "petite" mutation (rho-) has been reported (4-6). Removal ofcrosslinks has been described in Escherichia coli (7-10) and in mammalian cells in culture (11,12). In E. coli, the excision and the recombinational repair pathways are involved in the restitution of an intact genome from DNA containing psoralen photoproducts (7,9,10). In eukaryotes, limited information is available on the molecular steps in relation to the genetic control of repair of crosslinks in DNA. On the basis of sensitivity to cell killing in rad mutants compared to the wild type, it was demonstrated that in the yeast Saccharomyces cerevisiae, in addition to the excision (rad3 type), the error-prone (rad6 type), and the DNA double-strand-break (rad52) repair pathways (5, 13), some repair steps controlled by a class of genes designated pso (14) also are implicated in the repair of this last type of lesion.In this report, we determined the proportion of 8-(OMe)Ps photoinduced crosslinks as a function ofdose and followed their fate in nuclear and mtDNA in a normal strain. The influence of the growth phases, known to modify the lethal response (15), on crosslink removal was studied. An excision-deficient mutant (rad3) and one of the pso mutants (pso2) defective in the G2-phase repai...

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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.

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.