The Formulation of the Checkpoint Concept

T. A. Weinert and L. H. Hartwell, 1988, Science 241:317

Background

Hartwell and colleagues identified temperature-sensitive mutants that arrest in specific stages of the cell cycle in the budding yeast Saccharomyces cerevisiae. He called these mutants cdc mutants. Their subsequent characterization has profoundly shaped our understanding of the eukaryotic cell cycle. It was the characterization of one of these cdc mutants, cdc13, that led Ted Weinert and Lee Hartwell to formulate the checkpoint pathway concept.

The Experiment

The Cdc13 protein is required for telomere replication, and in its absence, large stretches of incompletely replicated telomeric DNA persist in cells. Cells carrying a temperature-sensitive allele of the cdc13 gene as the sole source of Cdc13 arrest with a G₂ DNA content when shifted to the restrictive temperature (Figure 1a). This arrest is indicative of a defect in late S phase, or entry into mitosis. When the cells are returned to the permissive temperature, the temperature-sensitive protein is functional again, and cells continue to proliferate. Thus, although the cdc13 mutant cells failed to divide at the restrictive temperature, they retained their viability and so were able to resume proliferation once they were returned to the permissive temperature at which the temperature-sensitive Cdc13 protein was functional again.

To characterize the cdc13 mutant in more detail, Weinert and Hartwell examined the effects of introducing a second mutation in another gene, a deletion in the RAD9 gene (Weinert and Hartwell, 1988). The RAD9 gene is not essential for viability, but when it is deleted, cells are extremely sensitive to DNA-damaging agents such as x-rays. This mutation on its own does not affect the growth of cells at any temperature, but it had a dramatic effect on cdc13 mutants. When the researchers examined the cdc13/rad9Δ double mutant at the restrictive temperature, they observed that the mutant no longer arrested in G₂; instead, the cells continued to divide for a few divisions (Figure 1b). When they returned these cells to the permissive temperature, the double mutant failed to resume proliferation. This observation indicated that while the cells continued to divide a few times at the restrictive temperature, they lost viability.

Weinert and Hartwell proposed the following explanation for this observation: the cdc13 mutants arrest at the restrictive temperature because they harbor incompletely replicated DNA. This damaged DNA signals the cell to arrest cell cycle progression and induce repair of the damage because mitosis of cells with damaged DNA would almost certainly lead to cell death. The RAD9 gene is part of the machinery that conveys this “halt cell cycle progression” signal. In cells lacking Rad9, this signal does not work, and cells undergo mitosis despite incompletely replicated DNA, which kills the cells. Weinert and Hartwell called this surveillance mechanism a checkpoint pathway.

Discussion

The checkpoint pathway concept had a profound effect not only on cell cycle research, but on all biological research. We now know that cell cycle progression is monitored by multiple surveillance mechanisms that ensure that cells grow to the appropriate size before they begin to divide, that their chromosomes are attached correctly to the spindle before they commence chromosome condensation, and that the nucleus is correctly positioned in the cell before they undergo cytokinesis. Checkpoint pathways also play critical roles during development. They ensure that subsequent developmental steps are not initiated before a prior step has been completed.

Importantly, checkpoint pathways, especially the DNA damage checkpoint pathway, are often inactivated in cancer, illustrating their importance for cellular and organismal function. The checkpoint concept is a powerful example of how studies in model organisms provide fundamental insights into all of biology as well as important insights into human diseases.