Condensins I and II are multisubunit complexes that play essential yet distinct functions in chromosome condensation and segregation in mitosis. Unlike condensin I, condensin II localizes to the nucleus during interphase, but it remains poorly understood what functions condensin II might have before mitotic entry. Here, we report that condensin II changes its chromatin-binding property during S phase. Remarkably, advanced premature chromosome condensation (PCC) assays enabled us to visualize condensin II forming “sister axes” in replicated regions of chromosomes in S phase cells. Depletion of condensin II compromised PCC-driven sister chromatid resolution during S phase. Moreover, fluorescence in situ hybridization assays revealed that condensin II, but not condensin I, promotes disjoining duplicated chromosomal loci during S phase. Application of mild replicative stress partially impaired this process and further exacerbated phenotypes arising from condensin II depletion. Our results suggest that condensin II initiates structural reorganization of duplicated chromosomes during S phase to prepare for their proper condensation and segregation in mitosis.

Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature chromosome condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of chromosome condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II by competing for its chromosomal binding sites in vitro. This simple and powerful assay allows us to dissect mutations causing primary microcephaly in vivo and evolutionary substitutions among different species. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate chromosome condensation and shaping.