Temporal and spatial coordination of DNA segregation and cell division in an archaeon Cells must coordinate DNA segregation with cytokinesis to ensure that each daughter cell inherits a complete genome. Here, we explore how DNA segregation and division are mechanistically coupled in archaeal relatives of eukaryotes, which lack Cyclin-dependent kinase (CDK)/Cyclins. Using live cell imaging, we first describe the series of sequential changes in DNA organization that accompany cell division in Sulfolobus, which computational modeling shows likely aid genome segregation. Through a perturbation analysis we identify a regulatory checkpoint which ensures that the compaction of the genome into two spatially segregated nucleoids only occurs once cells have assembled a division ring—which also defines the axis of DNA segregation. Finally, we show that DNA compaction and segregation depend, in part, on a ParA homologue, SegA, and its partner SegB, whose absence leads to bridging DNA. Taken together, these data show how regulatory checkpoints like those operating in eukaryotes aid high-fidelity division in an archaeon. 122 42 e2513939122 e2513939122 National Academy of Sciences application/pdf 1