Cellular functions, such as signal transmission, are carried out by 'modules' made up of many species of interacting molecules. Understanding how modules work has depended on combining phenomenological analysis with molecular studies. General principles that govern the structure and behaviour of modules may be discovered with help from synthetic sciences such as engineering and computer science, from stronger interactions between experiment and theory in cell biology, and from an appreciation of evolutionary constraints.

The events of the cell cycle of most organisms are ordered into dependent pathways in which the initiation of late events is dependent on the completion of early events. In eukaryotes, for example, mitosis is dependent on the completion of DNA synthesis. Some dependencies can be relieved by mutation (mitosis may then occur before completion of DNA synthesis), suggesting that the dependency is due to a control mechanism and not an intrinsic feature of the events themselves. Control mechanisms enforcing dependency in the cell cycle are here called checkpoints. Elimination of checkpoints may result in cell death, infidelity in the distribution of chromosomes or other organelles, or increased susceptibility to environmental perturbations such as DNA damaging agents. It appears that some checkpoints are eliminated during the early embryonic development of some organisms; this fact may pose special problems for the fidelity of embryonic cell division.

Multiple genetic changes occur during the evolution of normal cells into cancer cells. This evolution is facilitated in cancer cells by loss of fidelity in the processes that replicate, repair, and segregate the genome. Recent advances in our understanding of the cell cycle reveal how fidelity is normally achieved by the coordinated activity of cyclin-dependent kinases, checkpoint controls, and repair pathways and how this fidelity can be abrogated by specific genetic changes. These insights suggest molecular mechanisms for cellular transformation and may help to identify potential targets for improved cancer therapies.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G 2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the doublestrand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G 2 , these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.Many temperature-sensitive mutations that cause cells to arrest at specific stages in the cell cycle have been isolated in the yeast Saccharomyces cerevisiae (49). Mutations in four genes, CDC2, -9, -13, and -17, result in arrest in late S or G 2 phase. Cells defective for these genes probably arrest because of defects in the completion of DNA replication. CDC2, -9, and -17 are all known to encode components of the DNA replication machinery: CDC2 and CDC17 code for DNA polymerases (5, 13), whereas CDC9 codes for DNA ligase (36). Furthermore, mutations in all four genes induce high levels of mitotic recombination at the restrictive temperature, a result consistent with the presence of DNA lesions that remain after incomplete DNA replication (25). RAD9 is part of a system of genes that detects the presence of X-ray-induced DNA damage and stops the cell cycle in G 2 to allow time for repair before the cell enters mitosis (64). The RAD9 checkpoint may also recognize spontaneous damage and/or incomplete DNA replication, since rad9 deletion mutants experience an elevated level of chromosome loss (65). In a rad9 mutant background, cells with mutations in any one of these four CDC genes fail to arrest in G 2 ; instead they proceed into the next cell cycle. This result has been interpreted to mean that mutations in all four genes leave damage after DNA replication and that this damage produces a G 2 arrest mediated by the RAD9 pathway (66). In a RAD9 background, these mutants retain viability for a few hours at the restrictive temperature because mitosis in the presence of DNA damage is prevented; however, in a rad9 background, all four mutant strains die rapidly.Unlike those of CDC2, CDC9, and CDC17, the function of CDC13 is not yet understood. The sequence, reported here, for the CDC13 gene does not suggest a biochemical function. The DNA lesions left in cdc13 mutants display two properties in a rad9 background that distinguish them from the lesions left in cdc2, cdc9, and cdc17 mutants. First, the maxi...

In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. 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 Gz-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MECl and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13-, cdcl3 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdcl3 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-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 in different stages of the cell cycle at the restrictive temperature and 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/or DNA damage and cause arrest of cells before mitosis.

Early detection represents one of the most promising approaches to reducing the growing cancer burden. It already has a key role in the management of cervical and breast cancer, and is likely to become more important in the control of colorectal, prostate and lung cancer. Early-detection research has recently been revitalized by the advent of novel molecular technologies that can identify cellular changes at the level of the genome or proteome, but how can we harness these new technologies to develop effective and practical screening tests?

Cell division is arrested in many organisms in response to DNA damage. Examinations of the genetic basis for this response in the yeast Saccharomyces cerevisiae indicate that the RAD9 gene product is essential for arrest of cell division induced by DNA damage. Wild-type haploid cells irradiated with x-rays either arrest or delay cell division in the G2 phase of the cell cycle. Irradiated G1 and M phase haploid cells arrest irreversibly in G2 and die, whereas irradiated G2 phase haploid cells delay in G2 for a time proportional to the extent of damage before resuming cell division. In contrast, irradiated rad9 cells in any phase of the cycle do not delay cell division in G2, but continue to divide for several generations and die. However, efficient DNA repair can occur in irradiated rad9 cells if irradiated cells are blocked for several hours in G2 by treatment with a microtubule poison. The RAD9-dependent response detects potentially lethal DNA damage and causes arrest of cells in G2 until such damage is repaired.

Abstract. Time-lapse photomicroscopy has been utilized to detect temperature-sensitive yeast mutants that are defective in gene functions needed at specific stages of the cell-division cycle. This technique provides two types of information about a mutant: the time at which the defective gene function is normally performed, defined as the execution point, and the stage at which cells collect when the function is not performed, defined as the termination point.Mutants carrying lesions in three genes that control the cell-division cycle are described. All three genes, cdc-1, cdc-2, and cdc-3, execute early in the cell cycle at about the time of bud initiation, but differ in their termination points. Cells carrying the cdc-1 mutation terminate at the execution point, most cells ending up with a tiny bud that does not develop further. Cells carrying the cdc-2 mutation terminate at mitosis. Cells carrying the cdc-3 mutation are defective in cell separation but show no definite termination point since other processes of the cell cycle, such as bud initiation and nuclear division, continue despite the block in cell separation.