Type II topoisomerases are essential for resolving topologically entwined 
double-stranded DNA. Although anti-topoisomerase 2 (Top2) drugs are clinically 
important antibiotics and chemotherapies, to our knowledge, the mechanisms of 
cell killing by Top2 depletion and inactivation have never been directly 
compared. We show that depletion of Top2 protein from budding yeast cells 
prevents DNA decatenation during S phase. Cells complete DNA replication and 
enter the ensuing mitosis on schedule, suffering extensive chromosome 
missegregation. Cytokinesis through incompletely segregated chromosomes causes 
lethal DNA damage. By contrast, expression of catalytically inactive Top2 causes 
a stable G2 arrest requiring an intact DNA damage checkpoint. Checkpoint 
activation correlates with an inability to complete DNA replication, resulting 
in hypercatenated, gapped daughter DNA molecules. Thus, Top2 depletion and 
inactivation kill cells by different mechanisms, which has implications for 
understanding the nature of the catenation checkpoint, how DNA replication 
terminates, how anti-Top2 drugs work, and how new drugs might be designed.