Nieve Magaña-Schwencke

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.

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.

Biochemical analysis of damage induced in yeast by formaldehyde III. Repair of induced cross-links between DNA and proteins in the wild-type and in excision-deficient strains

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.

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.

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

SummaryThe 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 sublconed 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 tRAN 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.

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).

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.

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 recombination 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.

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 ydr013wΔ haploid cells were blocked after one or two cell cycles and presented heterogeneous bud sizes. The ydr021wΔ 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. Copyright