The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei‐41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the ‘lipid kinase’ subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei‐41. ATR can functionally complement esr1–1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.

During the cell cycle, DNA is replicated and segregated equally into two daughter cells. The DNA damage checkpoint ensures that DNA damage is repaired before mitosis is attempted. Genetic studies of the fission yeast Schizosaccharomyces pombe have identified two genes, rad24 and rad25, that are required for this checkpoint. These genes encode 14-3-3 protein homologs that together provide a function that is essential for cell proliferation. In addition, S. pombe rad24 null mutants, and to a lesser extent rad25 null mutants, enter mitosis prematurely, which indicates that 14-3-3 proteins have a role in determining the timing of mitosis.

The 14‐3‐3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin‐dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP‐ribosy‐lation of host proteins. In addition, a plant 14‐3‐3 protein is present in a G‐box DNA/protein‐binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14‐3‐3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14‐3‐3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted. The cDNA encoding a plant 14‐3‐3 protein under the control of the inducible GAL1 promoter complemented the double disruption. Transfer of the complemented double disruptant to a medium with glucose resulted in the appearance of a high percentage of large budded cells. After prolonged incubation, these cells became enlarged with irregular buds and chains of cells defective in cell‐cell separation became visible. These results suggest an essential role of the 14‐3‐3 proteins, possibly at a later stage of the yeast cell cycle.

The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

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