Topoisomerase IIα (topollα) and 13S condensin are both required for mitotic chromosome assembly. Here we show that they constitute the two main components of the chromosomal scaffold in histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. Topollα and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topollα and condensin generates a barber pole appearance of the scaffolding, where topollaα-and condensin-enriched "beads" alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatides; they lack this complex but contain a topollα-defined (-mediated?) scaffolding. Condensin associates only during the proto metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding.
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