A novel mechanism for chromatin bridge sensing through the abscission checkpoint in human cells

Eleni Petsalaki; Sofia Balafouti; George Zachos

Abstract:

Introduction

In the final stages of mitotic cell division, the narrow cytoplasmic canal that connects the two daughter cells is cut, and this is called abscission. However, sometimes chromatin bridges can arise during anaphase and persist in cytokinesis, and these are often the result of incomplete DNA replication or fusions of dicentric chromosomes. Chromatin bridges activate the abscission checkpoint, which is a mechanism that prolongs abscission in order to provide cells with enough time to resolve DNA bridges, and this has been linked to tumorigenesis. The abscission checkpoint prevents chromosome breakage if the cytoplasmic canal is cut, or tetraploidization in the case of furrow regression.

Methods

We performed point mutations in DNA constructs, reduced the levels of endogenous proteins with siRNAs, and used FISH and TUNEL. We also used confocal and time-lapse microscopy.

Results

We recently showed that the Mre11-Rad50-Nbs1 complex activates the ATM kinase, and this activates the Chk2 kinase in order to promote CPC localization to the midbody in cytokinesis with chromatin bridges. In this study, we show that Topoisomerase IIa (Top2a) localizes on DNA knots, which are regions of tangled DNA near the midbody, and creates abortive Top2ccs that are degraded in the proteasome. The degradation of Top2ccs is necessary for the localization of Rad17 on chromatin bridges. Rad17 then recruits the Mre11-Rad50-Nbs1 complex and activates downstream effector proteins to ensure the integrity of the chromatin bridges. Top2a-deficient cells exhibit reduced localization of the Mre11-Rad50-Nbs1 complex, Chk2, and the CPC complex and an increased frequency of broken DNA bridges. Interestingly, bridges derived from dicentric chromosomes do not possess knots or Top2ccs and are incapable of activating the abscission checkpoint.

Conclusions

We identified a new mechanism that cells use to recognize chromatin bridges during cytokinesis by creating abortive Top2ccs located on DNA knots to safeguard genome integrity and protect against tumorigenesis.