Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed “beans”) with condensin-positive central cores in its absence. The bean phenotype depends on the SMC ATPase cycle and can be reversed by subsequent addition of topo IIα. The HEAT repeat subunit CAP-D2, but not CAP-G, is essential for the bean formation. Notably, loop extrusion activities of the mutant complexes cannot explain the chromosomal defects they exhibit in Xenopus egg extracts, implying that a loop extrusion–independent mechanism contributes to condensin I–mediated chromosome assembly and shaping. We provide evidence that condensin–condensin interactions underlie these processes.

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis-specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis-specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

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