Following DNA damage, cells of the yeast Saccharomyces cerevisiae undergo a RAD9-dependent arrest in the G2 phase of the cell cycle. We are attempting to elucidate the molecular mechanism that allows DNA lesions to signal global cellular responses to DNA damage. The effects of an enzymatically induced double-strand break at a 45 bp YZ sequence (from MATYZ) in a nonyeast region of a dispensable single copy plasmid were examined. Induction of a persistent unrepaired break in trans results in nearly complete loss of plating efficiency even though the plasmid is dispensable. This loss of plating efficiency is partially dependent on the RAD9 gene product. Examination of single cells from rapidly cycling unsynchronized populations of RAD+ and rad9 cells following induction of the DSB indicated that over one-third of the cells (including both unbudded [G-1] and budded [S = G-2] cells) were inhibited from further cell division. The remaining cells of a RAD+ strain give rise primarily to microcolonies containing permanently arrested aberrantly shaped cells. Microcolonies were not observed in the rad9 background which accounts for the increased plating efficiency of this strain. This system provides a means for studying the signalling effects of a DNA lesion as well as designing strategies for modulating cell proliferation. In a study using the above methodology, we have investigated whether a site specific DSB can be used to physically map specific genes within the yeast genome. We have constructed and integrated LEU2YZ, URA3YZ and RNC1YZLEU2 targeting vectors into the yeast genome. Following integration, the yeast were transformed with a selectable plasmid containing the HO endonuclease gene under gal control Galactose induced a size specific chromosomal fragmentation at the LEU2YZ and URA3YZ integration sites on chromosomes III and V respectively corresponding to the known physical location of these genes. These results indicate that this approach can be used successfully to physically map cloned human or yeast genes within the yeast genome or upon YACs containing human DNA.
| Lewis, L Kevin; Karthikeyan, G; Cassiano, Jared et al. (2005) Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair. Nucleic Acids Res 33:4928-39 |
| Bennett, C B; Snipe, J R; Westmoreland, J W et al. (2001) SIR functions are required for the toleration of an unrepaired double-strand break in a dispensable yeast chromosome. Mol Cell Biol 21:5359-73 |
| Bennett, C B; Lewis, L K; Karthikeyan, G et al. (2001) Genes required for ionizing radiation resistance in yeast. Nat Genet 29:426-34 |
