The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide 
depletion by preventing replication fork collapse and the segregation of 
unreplicated chromosomes. Although the block to chromosome segregation has been 
thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 
mutants undergo cycles of spindle extension and collapse after hydroxyurea 
treatment that are distinct from anaphase cells. Furthermore, chromatid 
cohesion, whose dissolution triggers anaphase, is dispensable for S phase 
checkpoint arrest. Kinetochore-spindle attachments are required to prevent 
spindle extension during replication blocks, and chromosomes with two 
centromeres or an origin of replication juxtaposed to a centromere rescue the 
rad53 checkpoint defect. These observations suggest that checkpoint signaling is 
required to generate an inward force involved in maintaining preanaphase spindle 
integrity during DNA replication distress. We propose that by promoting 
replication fork integrity under these conditions Rad53 ensures centromere 
duplication. Replicating chromosomes can then bi-orient in a cohesin-independent 
manner to restrain untimely spindle extension.