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T A Weinert

Publications and source records attributed to T A Weinert.

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Dual cell cycle checkpoints sensitive to chromosome replication and DNA damage in the budding yeast Saccharomyces cerevisiae.

In eucaryotic cells chromosomes must be fully replicated and repaired before mitosis begins. Genetic studies indicate that this dependence of mitosis on completion of DNA replication and DNA repair derives from a negative control called a checkpoint which somehow checks for replication and DNA damage and blocks cell entry into mitosis. Here we summarize our current understanding of the genetic components of the cell cycle checkpoint in budding yeast. Mutants were identified and their phase and signal specificity tested primarily through interactions of the arrest-defective mutants with cell division cycle mutants. The results indicate that dual checkpoint controls exist in budding yeast, one control sensitive to inhibition of DNA replication (S-phase checkpoint), and a distinct but overlapping control sensitive to DNA repair (G2 checkpoint). Six genes are required for arrest in G2 phase after DNA damage (RAD9, RAD17, RAD24, MEC1, MEC2, and MEC3), and two of these are also essential for arrest in S phase when DNA replication is blocked (MEC1 and MEC2).

Cell Cycle

Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.

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.

Cell Cycle

Checkpoints: controls that ensure the order of cell cycle events.

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.

Animals

The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae.

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.

Cell Cycle

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

DNA Replication

Insertion sequence duplication in transpositional recombination.

Insertion sequences (IS) are discrete segments of DNA that can transpose from one genomic site to another and promote genetic rearrangements. A question that is central to understanding the mechanism of transpositional recombination is whether genetic rearrangements are accompanied by duplication of the IS that promotes them. Analysis of adjacent deletions mediated by IS903 provides the strongest evidence to date than any IS-mediated transpositional recombination can occur by an efficient replicative mechanism.

Base Sequence