Mutational analysis of the Drosophila CMG helicase reveals relationships among chromosome integrity and the maintenance of spindle and centrosome structure.

Abstract

The CMG (Cdc45-MCM-GINS) complex is a conserved helicase that plays an essential DNA unwinding function at replication forks. Here we analyzed the mitotic phenotypes caused in Drosophila by knockdown of Cdc45, Mcm5 and the four GINS genes (Sld5, Psf1, Psf2 and Psf3). Silencing of these genes resulted in virtually identical mitotic phenotypes. Brain cells from mutant and RNAi larvae showed severe defects in chromosome condensation, chromosome breakage and frequent polyploid mitotic figures. In addition, mutant cells showed reduced Cid (Cenp-A) incorporation at centromeres and strong alterations in spindle and centrosome structures. Our cytological and genetic analyses suggest that replication-related DNA damage and Cid-dependent centromere/kinetochore defects trigger the spindle assembly checkpoint (SAC) that arrests the cells in a prometaphase-like stage. The arrested cells undergo mitotic slippage accompanied by Cyclin B degradation, and eventually return to G1 giving rise to polyploid cells. Our analyses further suggest that during the prolonged prometaphase arrest both the centrosomes and the spindles undergo severe structural degeneration, and that the spindle defects are not the consequence of the aberrant centrosome behavior. Most studies on mitotic slippage have been carried out in cells exposed to anti-microtubule agents and could not address the behavior of the spindle. Conversely, our results illuminate the complex consequences of replication stress and reveal what happens to the mitotic apparatus during the prolonged SAC-induced mitotic arrest. Because prolonged mitosis is a common event in human cancers, our results could provide useful information for studies on cancer etiology and therapy.