Genome-Scale Genetic Interactions and Cell Imaging Confirm Cytokinesis as Deleterious to Transient Topoisomerase II Deficiency in Saccharomyces cerevisiae

Topoisomerase II (Top2) is an essential protein that resolves DNA catenations. When Top2 is inactivated, mitotic catastrophe results from massive entanglement of chromosomes. Top2 is also the target of many first-line anticancer drugs, the so-called Top2 poisons. Often, tumors become resistant to these drugs by acquiring hypomorphic mutations in the genes encoding Top2. Here, we have compared the cell cycle and nuclear segregation of two coisogenic Saccharomyces cerevisiae strains carrying top2 thermosensitive alleles that differ in their resistance to Top2 poisons: the broadly-used poison-sensitive top2-4 and the poison-resistant top2-5. Furthermore, we have performed genome-scale synthetic genetic array (SGA) analyses for both alleles under permissive conditions, chronic sublethal Top2 downregulation, and acute, yet transient, Top2 inactivation. We find that slowing down mitotic progression, especially at the time of execution of the mitotic exit network (MEN), protects against Top2 deficiency. In all conditions, genetic protection was stronger in top2-5; this correlated with cell biology experiments in this mutant, whereby we observed destabilization of both chromatin and ultrafine anaphase bridges by execution of MEN and cytokinesis. Interestingly, whereas transient inactivation of the critical MEN driver Cdc15 partly suppressed top2-5 lethality, this was not the case when earlier steps within anaphase were disrupted; i.e., top2-5 cdc14-1. We discuss the basis of this difference and suggest that accelerated progression through mitosis may be a therapeutic strategy to hypersensitize cancer cells carrying hypomorphic mutations in TOP2.

. Examples of uncoupling between nuclear division and cell cycle in top2-ts mutants. HTA2-GFP top2-ts cells were filmed as in Figure 2A. A. A cell in which a new bud (#) comes out from the mother before it had finished segregating the DNA with the first bud (observed in two top2-4 cells).
B. An example of the formation of a double CAB (arrowhead) that connects the mother cells with its two daughters (observed in one top2-5 cell). C. A nucleus trying to divide inside the mother cell, resulting in a temporarily binucleated single cell (*) that then fuses the split nuclei back into one single nuclear mass (observed in one top2-4 cell). The total number of cells filmed was 239 and include both top2-ts mutants as well as cells which were in either G1 or S phase (small bud) at the time of the temperature shift.

LEGENDS TO EXCEL FILES INCLUDED IN FILE S2.zip
File: SGAtool data_top2-4_TSv6_RT.xlsx Raw and processed SGA data of the permissive (25º) experiment where top2-4 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-4_TSv6_30C.xlsx Raw and processed SGA data of Top2 constant downregulation (30º) experiment where top2-4 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-4_TSv6_37Cx6h.xlsx Raw and processed SGA data of the transient Top2 inactivation (37º x 6h) experiment where top2-4 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-4_DMA_RT.xlsx Raw and processed SGA data of the permissive (25º) experiment where top2-4 was the query allele used against the collection of gene deletions.
File: SGAtool data_top2-4_DMA_30C.xlsx Raw and processed SGA data of Top2 constant downregulation (30º) experiment where top2-4 was the query allele used against the collection of gene deletions.
File: SGAtool data_top2-4_DMA_37Cx6h.xlsx Raw and processed SGA data of the transient Top2 inactivation (37º x 6h) experiment where top2-4 was the query allele used against the collection of gene deletions.
File: SGAtool data_top2-5_TSv6_RT.xlsx Raw and processed SGA data of the permissive (25º) experiment where top2-5 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-5_TSv6_30C.xlsx Raw and processed SGA data of Top2 constant downregulation (30º) experiment where top2-5 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-5_TSv6_37Cx6h.xlsx Raw and processed SGA data of the transient Top2 inactivation (37º x 6h) experiment where top2-5 was the query allele used against the collection of thermosensitive alleles.
File: SGAtool data_top2-5_DMA_RT.xlsx Raw and processed SGA data of the permissive (25º) experiment where top2-5 was the query allele used against the collection of gene deletions.