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
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.
University of Minnesota Ph.D. dissertation. August 2011. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Duncan J Clarke, PhD. 1 computer file (PDF) viii, 88 pages, appendix p. 86-88.
Smith, Katherine Laura.
Topoisomerase II: characterization of the Topoisomerase II checkpoint and the Classic top2-4 mutant allele..
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