Following chromosomal DNA damage (including DSBs), cells of the yeast Saccharomyces cerevisiae undergo a RAD9-dependent arrest in the G-2 phase of the cell cycle suggesting signalling between chromosomal lesions and cell cycling. To address the indirect """"""""global"""""""" effects of a DSB, we developed an inducible in vivo system for the production of a single-site specific DSB in a dispensable extrachromosomal plasmid. Nonswitching haploid RAD9+ and rad9-delta S. cerevisiae strains, each carrying a low copy CEN Plasmid with the HO endonuclease fused to the Gal1-10 promoter were transformed with a second selectable CEN plasmid containing a 45 bp MATYZ junction flanked between blocks of nonhomologous nonyeast, Galactose induction of HO endonuclease produced a unique and persistent site specific DSB at the YZ junction. This resulted in an immediate and permanent RAD9-independent arrest in over one third of the cells in a stage other than G-2. The remaining cells of a RAD+ strain gave rise primarily to microcolonies containing permanently arrested cells. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the trans-signalling effects of a DNA lesion as well as designing strategies for modulating cell proliferation and the indirect effects of DNA damaging agents. In a related study we are investigating whether a site specific DSB can be used to physically map specific genes within the yeast genome. We have constructed LEU2YZ and LEU2YZRNC1 targeting vectors. Following targeted integration the strains were transformed with a second selectable plasmid containing the HO endonuclease under gal control. Fragmentation of chromosome III at the LEU2 YZ integration site was observed following galactose induction. These results suggest that integration of cloned unmapped genes with a closely linked YZ junction can be physically mapped using this strategy. We anticipate that this approach will provide the basis for direct physical mapping of cloned genes.
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| Bennett, C B; Lewis, L K; Karthikeyan, G et al. (2001) Genes required for ionizing radiation resistance in yeast. Nat Genet 29:426-34 |
