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.

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.

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.

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.

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.