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In eukaryotic cells, checkpoint genes cause arrest of cell division when DNA is damaged or when DNA replication is blocked. In this study of budding yeast checkpoint genes, we identify and characterize another role for these checkpoint genes after DNA damage-transcriptional induction of genes. We found that three checkpoint genes (of six genes tested) have strong and distinct roles in transcriptional induction in four distinct pathways of regulation (each defined by induction of specific genes). MEC1 mediates the response in three transcriptional pathways, RAD53 mediates two of these pathways, and RAD17 mediates but a single pathway. The three other checkpoint genes (including RAD9) have small (twofold) but significant roles in transcriptional induction in all pathways. One of the pathways that we identify here leads to induction of MEC1 and RAD53 checkpoint genes themselves. This suggests a positive feedback circuit that may increase the cell's ability to respond to DNA damage. We make two primary conclusions from these studies. First, MEC1 appears to be the key regulator because it is required for all responses (both transcriptional and cell cycle arrest), while other genes serve only a subset of these responses. Second, the two types of responses, transcriptional induction and cell cycle arrest, appear distinct because both require MEC1 yet only cell cycle arrest requires RAD9. These and other results were used to formulate a working model of checkpoint gene function that accounts for roles of different checkpoint genes in different responses and after different types of damage. The conclusion that the yeast MEC1 gene is a key regulator also has implications for the role of a putative human homologue, the ATM gene.

In eucaryotic cells, incompletely replicated or damaged chromosomes induce cell cycle arrest in G2 before mitosis, and in the yeast Saccharomyces cerevisiae the RAD9 gene is essential for the cell cycle arrest (T.A. Weinert and L. H. Hartwell, Science 241:317-322, 1988). In this report, we extend the analysis of RAD9-dependent cell cycle control. We found that both induction of RAD9-dependent arrest in G2 and recovery from arrest could occur in the presence of the protein synthesis inhibitor cycloheximide, showing that the mechanism of RAD9-dependent control involves a posttranslational mechanism(s). We have isolated and determined the DNA sequence of the RAD9 gene, confirming the DNA sequence reported previously (R. H. Schiestl, P. Reynolds, S. Prakash, and L. Prakash, Mol. Cell. Biol. 9:1882-1886, 1989). The predicted protein sequence for the Rad9 protein bears no similarity to sequences of known proteins. We also found that synthesis of the RAD9 transcript in the cell cycle was constitutive and not induced by X-irradiation. We constructed yeast cells containing a complete deletion of the RAD9 gene; the rad9 null mutants were viable, sensitive to X- and UV irradiation, and defective for cell cycle arrest after DNA damage. Although Rad+ and rad9 delta cells had similar growth rates and cell cycle kinetics in unirradiated cells, the spontaneous rate of chromosome loss (in unirradiated cells) was elevated 7- to 21-fold in rad9 delta cells. These studies show that in the presence of induced or endogenous DNA damage, RAD9 is a negative regulator that inhibits progression from G2 in order to preserve cell viability and to maintain the fidelity of chromosome transmission.