Monitoring the DNA Topoisomerase II Checkpoint in Saccharomyces cerevisiae.

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control which responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.

Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation.

By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.

Assaying topoisomerase II checkpoints in yeast.

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations and aberrant DNA condensation during mitosis. Recently, novel top2 alleles in the yeast Saccharomyces cerevisiae revealed a checkpoint control that responds to perturbed Topo II activity. Described in this chapter are protocols for assaying the phenotypes seen in top2 mutants on a cell biological basis in live cells: activation of the Topo II checkpoint using spindle morphology, chromosome condensation using fluorescently labeled chromosomal loci, and cell cycle progression by flow cytometry. Further characterization of this novel checkpoint is warranted so that we can further our understanding of the cell cycle, genomic stability, and the possibility of identifying novel drug targets.

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae.

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.

Topoisomerase II checkpoints: universal mechanisms that regulate mitosis.

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin.

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.