Condensin ATPase motifs contribute differentially to the maintenance of chromosome morphology and genome stability.

Effective transfer of genetic information during cell division requires a major reorganization of chromosome structure. This process is triggered by condensin, a conserved pentameric ATPase essential for chromosome condensation. How condensin harnesses the energy of ATP hydrolysis to promote chromatin reorganization is unknown. To address this issue, we performed a genetic screen specifically focused on the ATPase domain of Smc4, a core subunit of condensin. Our screen identified mutational hotspots that impair condensin's ability to condense chromosomes to various degrees. These mutations have distinct effects on viability, genome stability, and chromosome morphology, revealing unique thresholds for condensin enzymatic activity in the execution of its cellular functions. Biochemical analyses indicate that inactivation of Smc4 ATPase activity can result in cell lethality because it favors a specific configuration of condensin that locks ATP in the enzyme. Together, our results provide critical insights into the mechanism used by condensin to harness the energy of ATP hydrolysis for the compaction of chromatin.