Following DNA damage, eukaryotic cells undergo cell cycle arrest at both the G1-~S and G2-~M phases of the cell cycle. In the yeast Saccharomyces cerevisiae the arrest at G2 requires the RAD9 gene product. We are elucidating the molecular mechanism(s) that allow DNA lesions to signal global cellular responses affecting cell cycle progression, DNA repair and recombination. We developed a system that allows the induction of a single, site-specific double-strand break (DSB) that does not undergo repair in a dispensable plasmid. Induction of an unrepaired plasmid DSB results in almost complete loss of plating efficiency in a Rad+ repair background even when the plasmid is dispensable. Loss of plating efficiency is partially dependent on the RAD9 gene product. Examination of the growth of single cells from a rapidly cycling unsynchronized population of Rad+ and rad9delta indicates that 41-49% of the cells [including unbudded (G1) and budded (G1 + G2)] were inhibited from further division. The majority of the remaining cells in a Rad+ strain give rise to permanently arrested microcolonies (<30 cells) which are not observed in a rad9delata background. This system has allowed us to access the contribution that a number of candidate repair and/or recombination gene products play in this signalling pathway. Isogenic rad1delta, rad6delta, rad50delta, rad52delta, rnc1delta and rnc1delta rad52delta strains have been constructed and plating efficiency determined following induction of a plasmid DSB. All of these strains showed increases in survival when compared to Rad+. The rad1delta and rad50delta strains showed complete attenuation of the lethality induced by the plasmid DSB. The rnc1delta, rnc1deltarad52delta and rad6delta strains showed almost complete attenuation of lethality while the rad52delta strain showed only partial suppression of lethality. Thus, these results identify known DNA repair genes that also participate in a signally pathway leading to lethality. Using methodology for inducing a defined DSB, we have constructed and integrated the DSB site into the chromosomal genes LEU2, URA3, and RNC1 to physically map genes within the yeast genome. Furthermore, we have successfully integrated Alu vectors containing the DSB cut site into YACs, containing human DNA and mapped the integration position using Southern blotting of chromosomes following gel electrophesis. These results indicate that this approach can be used to successfully map cloned human or yeast genes within the yeast genome or within 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 |
