Dual Role of Topoisomerase II in Centromere Resolution and Aurora B Activity

Abstract

Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.

Author Summary

Successful cell division requires that chromosomes are properly condensed and that each sister chromatid is self-contained by the time the sister pairs are segregated into separate daughter cells. It is also essential that the kinetochores at the centromeres of each pair of sister chromatids bind microtubules from opposite spindle poles. Topoisomerase II is a highly conserved enzyme that removes interlinks from DNA and is known to be essential to proper chromosome segregation during cell division. In this work, we have used state-of-the-art four-dimensional fluorescent microscopy to follow progression through mitosis in living cells depleted of topoisomerase II. We find that when the enzyme is absent, the two sister centromeres do not separate, and chromosomes missegregate. Moreover, the inappropriate centromere structure that results prevents the correct activation of the Aurora B kinase, which forms part of a regulatory mechanism that monitors correct segregation of chromosomes; as a result, cells exit mitosis abnormally.

Abstract

Analysis of cells lacking topoisomerase II reveals that the enzyme has an essential role in the segregation of chromosomes, and specifically centromeres, at anaphase-telophase of mitosis: it prevents non-disjunction and allows activation of the Aurora B kinase, so as to correct improper attachments between microtubules and the kinetochore.