Coupling mitosis to S-phase

Abstract

In this thesis, we studied the structural and biochemical findings described in the introduction, from a more cell biological perspective. We investigated how replication licensing and mitosis might be linked. We also asked, how the spindle checkpoint and the APC/C can coordinate mitotic exit and DNA replication licensing in time and space. We identified how the spindle checkpoint controls geminin degradation in mitosis (chapter 2). Furthermore, we revealed how subsequent activation of APC/C co-activators may determine a time-window for replication licensing around anaphase (chapter 2). Also, we set out to unravel the paradoxical positive role of geminin in replication licensing, showing a role for geminin in stabilization of Cdt1 in prophase and prometaphase (chapter 2). In addition, we tried to reveal how Cdt1 protein levels may be controlled before, during and after mitosis (Addendum, chapter 2). In chapter 3, we find a new S-phase specific mechanism that controls Cdc6 protein levels, involving a previously unidentified destruction motif. We speculate that this motif, together with the APC/C-Cdh1 targeted Cdc6 KEN-box, and a Cdk1-dependent nuclear export mechanism that operates from late S-phase onwards, all contribute to preventing any re-licensing event during S-phase. Moreover, we tried to clarify the controversy over the role of progressive cyclin B1-Cdk1 inactivation in spindle checkpoint silencing, and reveal that amino acids around the cyclin B1 destruction box influence the efficiency of APC/C-Cdc20 dependent degradation (chapter 4). Finally, we discuss how the spindle checkpoint, cyclin B1-Cdk1 and geminin may work together to coordinate the coupling of mitotic exit to the onset of DNA replication in rapidly dividing cells, such as cancer cells (chapter 5). Finally, we discuss several controversies regarding the roles of licensing factors and inhibitors in preventing re-licensing events at the wrong time in the cell cycle (chapter 5).