Functional interplay between condensin I and topoisomerase IIα in single-molecule DNA compaction

Abstract Condensin I and topoisomerase IIα (topo IIα) are chromosomal ATPases essential for mitotic chromosome assembly. Mechanistically how the two ATPases cooperate to assemble mitotic chromosomes remains unknown. Here we use total internal reflection fluorescence microscopy to analyze the interplay between condensin I and topo IIα at single-molecule resolution. As observed in previous studies, condensin I alone predominantly forms DNA loops in an ATP-dependent manner. However, when topo IIα is included in the reaction, condensin I forms stable compact structures (termed “lumps”) in a manner dependent on the C-terminal domain of topo IIα. Each of the stable lumps contains a single condensin I complex and a single topo IIα dimer. Remarkably, we find that topo IIα, when catalytically active, renders the lumps resistant to protease treatment. Several lines of evidence show that the protease-resistant lumps contain knotted DNA. A mutant condensin I complex defective in ATP hydrolysis, together with topo IIα, forms smaller lumps in which the probability of DNA knotting is greatly reduced. Our results demonstrate how topo IIα-mediated strand passage is coupled with condensin I-mediated loop extrusion to generate a compact DNA structure. Together with recent studies, we discuss the functional implications of these observations in mitotic chromosome assembly and stabilization. Competing Interest Statement The authors have declared no competing interest.