Leland H. Hartwell

WHEN CHECKPOINTS FAIL

We would like to thank members of the Hartwell laboratory, especially Eric Foss, members of the Seattle project, Jim Roberts, Andrew Murray, and an annonymous reviewer for helpful comments on the manuscript. A. G. P. was supported by a Merck Distinguished Fellow Award and an MSTP training grant from the NIH. D. P. T. was supported by a fellowship from the Jane Coffin Childs Memorial Fund and an NIH training program in Cancer Research CA09437. L. H. H. is a Research Professor of the American Cancer Society.

Coordination of growth with cell division in the yeast Saccharomyces cerevisiae

Abstract Yeast cell growth and cell division are normally coordinated. The mechanism of this coordination can be examined by attempting to dissociate the two processes. We have arrested division (with the use of temperature-sensitive cell division cycle mutants) and observed the effect of this arrest on growth, and we have limited growth (by nutritional deprivation) and observed the effect of this limitation on division. Mutants blocked at various stages of the cell cycle were able to continue growth, as evidenced by increases in cell volume, mass, and protein content. Cells that had initiated cell cycles were able to complete their cycles and arrest in G1 even when growth was very severely restricted. Under these conditions the daughter cells produced were abnormally small. Such abnormally small cells did not initiate new cell cycles (i.e., did not bud or complete any of the three known gene-controlled steps ( cdc 28, cdc 4, cdc 7) in G1) after nutrients were restored until growth to a critical size had occurred. We have also prepared abnormally large cells (by arresting division temporarily with the appropriate mating pheromone); when such cells were allowed to bud, the buds produced were much smaller than the mother cells when cytokinesis occurred. We propose that the normal coordination of cell growth with cell division is a consequence of the following two relationships. (1) Growth, rather than progress through the DNA-division cycle, is normally rate-limiting for cell proliferation. (2) A specific early event in G1, at or before the event controlled by the cdc 28 gene product, cannot be completed until a critical size is attained.

A checkpoint regulates the rate of progression through S phase in S. cerevisiae in Response to DNA damage

We demonstrate that in S. cerevisiae the rate of ongoing S phase is slowed when the DNA is subjected to alkylation. Slowing of replication is dependent on the MEC1 and RAD53 genes, indicating that lesions alone do not slow replication in vivo and that the slowing is an active process. While it has been shown that a MEC1- and RAD53-dependent checkpoint responds to blocked replication or DNA damage by inhibiting the onset of mitosis, we demonstrate that this checkpoint must also have an additional target within S phase that controls replication rate. MEC1 is a homolog of the human ATM gene, which is mutated in ataxia telangiectasia (AT) patients. Like mec1 yeast, AT cells are characterized by damage-resistant DNA synthesis, highlighting the congruence of the yeast and mammalian systems.

Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis

Temperature-sensitive mutations in one gene (cdc1) of Saccharomyces cerevisiae confer a defect in bud emergence. Asynchronous cultures of cells defective in cdc1 collect uniformly as unbudded cells (or cells with very tiny buds) following a shift from the permissive to the restrictive temperature. Studies with synchronous cultures demonstrate that the thermolabile product of cdc1 completes its function (the execution point) for bud emergence at the time of bud emergence (0.2 fractions of a cell cycle). When this function is not completed at the restrictive temperature. cells complete DNA replication but do not undergo nuclear division. Temperature-sensitive mutations in four genes (cdc3, 10, 11, and 12) result in a defect in cytokinesis. At the restrictive temperature these mutant strains develop multiple elongated buds that do not separate from the parent cell. An assay for cytokinesis in yeast was developed and the mutants were shown not to have completed this process. The mutants undergo several rounds of DNA replication and nuclear division at the restrictive temperature and hence become multinucleate. The execution points for these gene products were determined in synchrony experiments to be at approx. 0.08 (cdc11), 0.27 (cdc3) and 0.58 (cdc10) fractions of a cell cycle. Cytokinesis takes place at 0.9 fractions of a cell cycle. I conclude from these observations that bud emergence is not a necessary prerequisite for the completion of DNA replication but is apparently necessary for nuclear division. Cytokinesis and cell separation are not necessary prerequisites for bud emergence, DNA replication, or nuclear division.

The Saccharomyces cerevisiae Cell Cycle

INTRODUCTION The cell cycle is the process of vegetative (asexual) cellular reproduction; in a normal cell cycle, one cell gives rise to two cells that are genetically identical to the original cell. Questions about the cell cycle can be conveniently divided into two categories. First, one can ask how a cell carries out a cell cycle, once it has undertaken to do so. Into this category fall questions about the morphological and biochemical aspects of cell-cycle events and about the mechanisms that ensure their temporal and functional coordination. Second, one can ask what determines when a cell will undertake a cell cycle, or how the overall control of cell proliferation is achieved. Into this category fall questions about the coordination of successive cell cycles, the coordination of growth with division, the coordination of cell proliferation with the availability of essential nutrients, and the selection of developmental alternatives. In the text that follows, we consider these two categories of questions in turn. Our bibliography is intended more as a guide to the literature than as a historically accurate record of the development of the field; we apologize to the earlier workers whose contributions thus get less explicit credit than they deserve. HOW DOES A CELL CARRY OUT A CELL CYCLE? As has often been noted, successful completion of a cell cycle requires a cell to integrate the processes that duplicate the cellular material with the processes that partition the duplicated material into two viable daughter cells. Another useful formulation of the...

CDC5 and CKII Control Adaptation to the Yeast DNA Damage Checkpoint

A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint. If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present. Here, we report the identification of two adaptation-defective mutants that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome. This adaptation-defective phenotype was entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest. We show that one mutation resides in CDC5, which encodes a polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II.

Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis

Abstract Four steps are known to be required for the initiation of DNA synthesis in Saccharomyces cerevisiae. Three of these are mediated by the products of genes cdc 4, 7, and 28 and the fourth is identified by the inhibition exerted on haploid α cells by the mating pheromone, α factor. These four steps have been ordered by a combination of two methods and found to be: initiation of DNA synthesis The two sequencing methods are described in detail. Experiments involving the shift of mutant cells from the restrictive to the permissive temperature in the presence of cycloheximide demonstrated that the protein synthesis requirement for yeast DNA replication can be completed before the cdc 7-mediated step.

Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible sex factor

Abstract A diffusible substance, α factor, is produced constitutively by haploid yeast cells of α mating type and this factor specifically inhibits the division of a mating type cells. Experiments are presented which demonstrate that α factor arrests a cells as unbudded, mononucleate cells prior to the initiation of DNA synthesis in the cell cycle. Studies with temperature-sensitive mutants defective in one of thirteen different cell cycle functions suggest that although arrested a cells continue to enlarge they do not perform functions required for the next cell cycle. The arrest is reversible and a partially synchronized round of DNA replication is observed upon removal of α factor from arrested cells. We propose that this factor is one element of a regulatory system that functions to assure the synchronization of a and α haploid cell cycles prior to conjugation.

Genetic control of the cell division cycle in yeast: II. Genes controlling DNA replication and its initiation☆☆☆

Temperature-sensitive mutations occurring in two unlinked complementation groups, cdc4 and cdc8, are recessive and result in a defect in DNA replication at the restrictive temperature. Results obtained with synchronous cultures suggest that cdc4 functions in the initiation of DNA replication and cdc8 functions in the propagation of DNA replication. From the behavior of mutant strains carrying lesions in cdc4, or in cdc8, or in both genes it is concluded that: (1) nuclear division and cell separation in yeast are dependent upon prior DNA replication; (2) a cellular clock controls bud initiation and the running of this clock is independent of the other events in the cycle, DNA replication, nuclear division and cell separation; (3) premature bud initiation is normally prevented as a consequence of the successful initiation of DNA replication.

Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission

To identify gene products that function stoichiometrically in mitotic chromosome transmission, genes were cloned on high copy number plasmids and transformed into yeast cells, and the transformants were examined for an increase in the frequency of mitotic chromosome loss or recombination resulting from the gene imbalance. When either pair of the yeast histone genes H2A and H2B, or H3 and H4 was present on high copy number plasmids, both chromosomes V and VII exhibited an increased frequency of chromosome loss. The rate of chromosome loss was not elevated when the histone genes were present on single copy plasmids, when their transcription from high copy plasmids was repressed, or when frame-shift mutations were present in the coding sequence. This method for the identification of genes circumvents some of the limitations of traditional mutational analysis and yields the cloned gene.