MITOTIC CHECKPOINT GENES IN BUDDING YEAST AND THE DEPENDENCE OF MITOSIS ON DNA-REPLICATION AND REPAIR

In eukaryotes a cell-cycle control termed a checkpoint causes arrest i n the S or G2 phases when chromosomes are incompletely replicated or d amaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage . Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The G2-specific genes include MEC3 (for mitosis entry chec kpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MEC1 and MEC2. The MEC2 gene proves to be identi cal to the RAD53 gene. Checkpoint mutants were identified by their int eractions with a temperature-sensitive allele of the cell division cyc le gene CDC13; cdc13 mutants arrested in G2 and survived at the restri ctive temperature, whereas all cdc13 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The c ell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest i n different stages of the cell cycle at the restrictive temperature an d by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/o r DNA damage and cause arrest of cells before mitosis.