DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior

Abstract

The DNA replication checkpoint maintains replication fork integrity and prevents chromosome segregation during replication stresses. Mec1 and Rad53 (human ATM/ATR- and Chk2-like kinases, respectively) are critical effectors of this pathway in yeast. When treated with replication inhibitors, checkpoint-deficient mec1 or rad53 mutant fails to maintain replication fork integrity and proceeds to partition unreplicated chromosomes. We show that this unnatural chromosome segregation requires neither the onset of mitosis nor APC activation, cohesin cleavage, or biorientation of kinetochores. Instead, the checkpoint deficiency leads to deregulation of microtubule-associated proteins Cin8 and Stu2, which, in the absence of both chromosome cohesion and bipolar attachment of kinetochores to microtubules, induce untimely spindle elongation, causing premature chromosome separation. The checkpoint's ability to prevent nuclear division is abolished by combined deficiency of microtubule-destabilizing motor Kip3 and Mad2 functions. Thus, the DNA replication checkpoint prevents precocious chromosome segregation, not by inhibiting entry into mitosis as widely believed, but by directly regulating spindle dynamics.