Key events in progression through the cell cycle are monitored through a series of checkpoints. Checkpoints assess the integrity of critical events during the cell cycle, such as the completion of DNA replication, and the presence of an appropriate mitotic spindle63
. Since topoisomerase II carries out a reaction that is essential for chromosome separation at mitosis, a plausible hypothesis is that cells can monitor the successful completion of topoisomerase II decatenation, and arrest cell cycle progression if decatenation (or chromosome condensation) is incomplete. Early studies using S. cerevisiae
and S. pombe
argued against this possibility, since conditional top2 mutants showed minimal cell cycle delay, and instead accumulated broken chromosomes at the time of mitosis 24,64-66
. Topoisomerase II poisons generate DNA damage, in addition to inhibition of enzyme activity, and would be expected to delay cell cycle progression due to DNA damage checkpoints.67-69
The demonstration by Andoh and colleagues that bisdioxopiperazines were specific catalytic inhibitors of eukaryotic topoisomerase II70
allowed a test in mammalian cells for the presence of a checkpoint for topoisomerase II function. Downes and colleagues found that bisdioxopiperazines such as ICRF-187 and ICRF-193 were able to elicit a caffeine sensitive delay of entry into mitosis71
. Subsequent work using ICRF-187 demonstrated a mitotic delay that was dependent on ATR and BRCA172
, but apparently independent of both DNA damage checkpoints and the spindle checkpoint73
. As carefully noted by Downes and colleagues, the checkpoint they identified depended on the properties of bisdioxopiperazines71
. Since they were able to show distinct differences between etoposide (as a standard Top2 poison) and ICRF-193, they concluded that the effects of ICRF-193 arose from a lack of Top2 activity.
An alternate approach to assessing whether cells monitor the completion of Top2 function is to completely deplete Top2 protein before mitosis. This has been done both in yeast and mammalian cells. As described above in the section on replication, a complete depletion of Top2 does not lead to a delay in mitosis in yeast cells25
, while expression of an inactive Top2 does lead to a mitotic delay. This finding is in agreement with a previous hypothesis that yeast cells carrying a temperature sensitive Top2 fails to arrest at mitosis because the presence of Top2 is needed to trigger the delay74
. However, the results of Diffley and colleagues suggest that the arrest seen in yeast is due to a problem with replication rather than decatenation. In mammalian cells, whether mitotic delay is induced by Top2α depletion is a point of controversy. Removal of Top2 using a conditional Tet-off system showed that loss of Top2 protein led to mitotic delay. By contrast, no delay was seen in cells depleted for Top2α using siRNA27
. In the latter experiments, loss of cell viability was clearly seen in cells depleted for Top2α. In experiments from other laboratories using siRNA directed against Top2α, no phenotype was observed presumably because the knockdown of Top2α was insufficient. Additional experiments, perhaps with primary cells will be useful in demarcating the types of cells that can carry out mitotic delay in response to insufficient Top2 activity. It is interesting to note that recent experiments indicate a lack of a mitotic delay induced by bisdioxopiperazines in embryonic stem cells and hematopoetic progenitor cells75,76
The checkpoint induced by bisdioxopiperazines is termed a decatenation checkpoint, but it would more accurately be termed a Top2 checkpoint, since there is no direct evidence that the mitotic delay monitors chromatid decatenation. It is not clear how the cell could assess sister chromatid catenation. The presence of catenanes is a property of a chromosomal domain, and it does not generate obvious local consequences. For example, cells might assess DNA supercoiling by “counting” crossing of the DNA double helix (formally writhe), but there is no obvious way to assess writhe that is specifically associated with catenanes. Assessment of catenation state may depend more on structural alterations, perhaps at centromeres.
There has been interest in determining whether the topoisomerase II checkpoint can be exploited for cancer therapy. A small molecule inhibitor of a bisdioxopiperazines induced checkpoint has been described, although the molecular target of the small molecule is unknown77
. This may be of particular use in concert with potent Top2 catalytic inhibitors. In any case, perturbing Top2 checkpoints are unlikely to be a major determinant of response to Top2 poisons, which depend more on DNA damage checkpoints for their efficacy.
Top2 is required in many biological contexts
The original impetus for studying Top2 came in part from the mysterious and complicated reactions the enzyme carries out. Therefore, early studies concentrated especially on the biochemical and structural aspects of the enzyme. These studies have now concluded with a detailed understanding of many critical issues of Top2 biochemistry. Top2 was expected to be important in chromosome replication and segregation, but the recent work suggests that decatenation of replicated chromosomes requires a precise choreography that includes regulating Top2 action both spatially and temporally. Importantly, cells may have the means of ensuring that these processes have occurred correctly, although how cells assess proper Top2 function remains unclear.
A recent surprise has been the unique roles played by Top2β. It was surprising that Top2β is specifically required in certain neuronal cells, and the finding that this enzyme is required for transcription of some genes will lead to further unappreciated biological roles for both Top2 isoforms. Although not discussed in detail here, Top2 has also been proposed to play roles in DNA repair78-80
, especially in the ability of DNA lesions such as abasic sites to generate enzyme mediated DNA damage81
. Other possible functions of Top2 will depend on a better understanding of the protein complexes that include Top2. As with other proteomic studies, identification of the relevant protein complexes is only the first step in understanding the relevant biological processes.
As described in the accompanying review, Top2 is especially relevant in cancer because it is the target of many active anti-cancer agents. At present, most drugs targeting Top2 kill cells by generating enzyme mediated DNA damage, rather than by inhibiting enzyme activity. The importance of Top2 in proliferating cells, as well as its roles in transcription, suggest that catalytic inhibition may also be a useful anti-cancer strategy. If this strategy proves useful, a more complete understanding of Top2 biological functions will be critical.
At a glance
- Type II topoisomerases change DNA topology by generating transient DNA double strand breaks. Type II topoisomerases are essential for all eukaryotic cells.
- Mammalian cells carry two Top2 isoforms, Top2α and Top2β. Top2α is essential for all cells, and is essential for separating replicated chromosomes. Top2β is required for normal development, but is dispensable in some cell types. Type II topoisomerases are required for other processes such as transcription, and the precise roles of the two isoforms in these processes are a subject of current studies.
- Type II topoisomerases use a “two gate” mechanism for carrying out topological changes in DNA. The enzyme requires ATP hydrolysis for its reaction. ATP hydrolysis is used for for conformational changes of the enzyme, and is not directly involved in DNA breakage or resealing.
- Structures of several domains of yeast Top2 have provided additional information about how the enzyme carries out its reactions. A recent structure of the breakage-reunion domain of yeast Top2 bound to DNA has shown that the enzyme induces a large bend in the DNA that is cleaved by the enzyme.
- Biological functions of Top2 isoforms are modulated be a variety of protein:protein interactions. Some of these interactions may affect enzyme activity, stability, and localization.
- Top2 activity is also modulated by post-translational modification. In addition to phosphorylation, a critical post-translational modification of Top2 is sumoylation. Failure to sumoylate Top2α or to remove the SUMO mopdification disrupts the ability of Top2α to separate replicated chromosomes.
- Top2β plays a key role in the survival of some neural cells. Top2β is important in transcriptional regulation, and it is likely that Top2β enzyme activity is specifically required.
- Some aspects of Top2 function during the cell cycle are monitored by checkpoints. It has been hypothesized that a major role of checkpoints are to monitor the completion of decatenation. If so, then Top2 dependent checkpoints may be critical for normal chromosome segregation and genome stability.