Title : A new sensing mechanism for chromatin bridges by the abscission checkpoint in human cancer cells
Abstract:
Chromatin bridges are DNA strings that link anaphase poles or daughter nuclei and are associated with carcinogenesis. DNA bridges occur from the separation of twisted, linked chromatin that is caused by problems in DNA replication or incomplete DNA decatenation.
In response to chromatin bridges, cells delay abscission by activating the abscission checkpoint which delays completion of cytokinesis in order to prevent chromatin breakage or tetraploidization by furrow regression. In mammalian cells, the abscission checkpoint is dependent on Aurora B kinase activation. We recently showed that in cytokinesis with chromatin bridges a biochemical pathway including MRN complex, ATM and Chk2 promotes INCENP localization to the midbody to delay abscission and prevent chromatin breakage.
In this study we used site directed mutagenesis, RNA silencing, time lapse and confocal microscopy, FISH, BrdU labeling, TUNEL assay and differential retention assay.
Here, we show that spontaneous or replication stress-induced chromatin bridges derived from catenated DNA exhibit “knots” of tangled, overtwisted DNA next to the midbody. Topoisomerase IIα (Top2a), an enzyme that relaxes supercoils and untangles catenated DNA forms irreversible Top2-DNA cleavage complexes (Top2ccs) on DNA knots. Inhibition of Top2a results in diminished localization of Rad17, MRN, ATM, Chk2 and CPC complex to the DNA bridges and induces chromatin bridge breakage. Furthermore, proteolytic degradation of Top2ccs is required for localization of Rad17 to the bridge DNA. In turn, Rad17 promotes recruitment of the MRN complex to DNA knots and downstream abscission checkpoint signaling to delay abscission and prevent chromatin bridge breakage in cytokinesis. In contrast, chromatin bridges generated by dicentric chromosomes do not exhibit DNA knots or Top2ccs next to the midbody, and fail to recruit Top2a, Rad17 and other downstream proteins and are unable to induce an abscission delay.
Our results describe a novel mechanism by which the abscission checkpoint detects chromatin bridges in human cells, through generation of irreversible Top2ccs on DNA knots. Because chromosomal instability is a major cause of cancer, identifying new genes that protect genome integrity is very important for cancer research.
