Topoisomerase II: characterization of the Topoisomerase II checkpoint and the Classic top2-4 mutant allele.

Abstract

Topoisomerase II (Topo II) is an essential, highly conserved enzyme that is a major component of mitotic chromosomes. Topo II functions as a homodimer to perform an ATP-dependent strand-passage reaction. In this reaction, Topo II transiently cuts one dsDNA molecule to allow the passage of a second dsDNA molecule. This cycle resolves knots in the DNA (catenanes) that form during replication and this resolution is essential for mitosis. Progression through the cell cycle in the absence of Topo II results in mitotic catastrophe (HOLM et al. 1985). The Clarke lab identified and provided genetic evidence that there is a Topo II checkpoint in budding yeast (ANDREWS et al. 2006). Failure of the Topo II checkpoint results in aneuploidy and reduced cell viability (ANDREWS et al. 2006). Unexpectedly, by using DNA damage checkpoint components Andrews et al. found that the Topo II checkpoint does not enforce a G2/M delay, but rather requires a subset of spindle checkpoint proteins. The Topo II checkpoint is different from the spindle checkpoint in that it is not activated when the spindle is damaged or when there is a lack of tension. Additionally, it does not delay cell cycle progression through Pds1-dependent inhibition of Esp1/separase (ANDREWS et al. 2006). Thus, the Topo II checkpoint is distinct from other known checkpoints. Due to the recent discovery of the Topo II checkpoint there is little known about what the checkpoint monitors or what is required for checkpoint signaling. To begin to answer these questions, we needed a way to look at a single cell cycle in the absence of Top2. Top2 is essential in cells; it cannot simply be deleted. Instead, a Top2 degron system was constructed in Saccharomyces cerevisiae. Using the Top2 degron we found that the checkpoint is dependent upon the presence of Top2 protein in the cell during the cell cycle. Under these conditions, the iv chromosomes should have abnormal condensation, kinetochore biorientation should be perturbed, and catenations should persist. Surprisingly, despite all this, the checkpoint is unable to sense the disturbance when there is no Top2 protein present. The Top2 degron system allows for the expression of Top2 mutants, which would normally be inviable. We found that mutants that affect the ability of Top2 to open its DNA gate to allow T-segment transport also activate the checkpoint. However, mutants that affect the strand-passage cycle at a later step do not activate the checkpoint. This is the first step in our mechanistic understanding of what the checkpoint monitors. In our attempt to begin to understand what is required for Topo II checkpoint activation, we found that the C-terminal tail of Top2 is necessary. Previous research has shown that this tail can be deleted without disturbing the strand-passage reaction or completion of the cell cycle (CARON et al. 1994 and JENSEN et al. 1996). However, our research shows that the checkpoint cannot be activated when the tail is deleted and that overexpression of the tail interferes with checkpoint activation. The tail is highly posttranslationally modified (ALGHISI et al. 1994 and BACHANT et al.2002) and most of these modifications have no known function. We hypothesize that the tail might be used to initiate Topo II checkpoint signaling and that it could provide a docking site for the initiating proteins. Finally, we more closely examine a well-used temperature sensitive mutant allele of top2, top2-4. This mutant was first identified and characterized by Holm et al. in 1985. It has since been extensively used as a null form of top2, when grown above its restrictive temperature. We found that this mutant does not behave as a null. Not only is the Top2- 4 protein stable above restrictive temperature, but it also has a dominant effect on the ability of Top2-B44 to activate the Topo II checkpoint.