The G2/M checkpoint senses DNA damage and through its signal transduction pathways arrests the cell cycle, providing time for DNA repair. In S. cerevisciae eight genes are known to subserve the G2/M checkpoint function. Earlier genetic screens were neither saturating of the genes detected nor successful in identifying all of the genes presently known to be required. Here, a visual morphologic screen is presented to identify novel G2/M checkpoint genes. To do so, a strain carrying cdc13 ts and cdc15 ts is mutagenized with EMS. At 36 degrees, cdc13 creates DNA damage by disrupting telomere metabolism. Checkpoint proficient cells arrest with medial nuclear division; checkpoint deficient ones progress through mitosis but are arrested post--anaphase by cdc15, and are thus easily recognizable. Colonies found deficient for the G2/M checkpoint will be characterized by complementation and allelism testing with known checkpoint genes. The remaining unknown mutations are grouped by complementation and the phenotypically strongest allele analyzed further. Mutations of cell cycle machinery genes, which also may be checkpoint deficient, will be identified by plasmid transformation. The remaining, novel genes will be cloned by plasmid library transformation and sequenced. Knockout mutations of the novel genes will be exploited in tests of genetic interaction (i.e., epistasis, synthetic lethality) with known checkpoint genes. Finding novel checkpoint genes, especially downstream components of the pathway may provide new insights into the mechanism of cell cycle arrest. Ultimately, checkpoint pathways may prove useful targets for therapeutic intervention in cancer, since many human cancers harbor checkpoint defects (e.g., mutations in p53 or ATM).
| Gardner, R; Putnam, C W; Weinert, T (1999) RAD53, DUN1 and PDS1 define two parallel G2/M checkpoint pathways in budding yeast. EMBO J 18:3173-85 |
