Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes

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(1)Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes Duursma, A.M.. Citation Duursma, A. M. (2008, May 14). Molecular dissection of Cdc6 and the miR-148 family : two stories with common themes. Retrieved from https://hdl.handle.net/1887/12848 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/12848. Note: To cite this publication please use the final published version (if applicable)..

(2) Chapter 3. CDK-dependent stabilization of Cdc6: Linking growth and stress signals to activation of DNA replication

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(7) CDK-dependent stabilization of Cdc6: Linking growth and stress signals to activation of DNA replication

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(13) %'$ Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G1 phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.. Origin determination # $ %$$&%'

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(103) CDK-dependent regulation of Cdc6. Nevertheless, Cdc6 failed to be recognized as a positive regulator of DNA replication. Interestingly, latest observations establish Cdc6 as the first example of a key replication initiation protein whose stability is increased by CDK phosphorylation. We recently observed that the licensing protein Cdc6 is phosphorylated and thereby stabilized by CDK2/cyclin E activity (Duursma and Agami, 2005). In particularly, phosphorylation of one amino acid (serine 54) protects Cdc6 from APCCdh1 mediated destruction. In line with our results, Mailand et al showed that phosphorylation of Cdc6 prevents it Cdh1dependent ubiquitination (Mailand and Diffley, 2005). The fact that Cdc6 stability is controlled by CDK2 implies regulation through the p53 pathway in stress responses. DNA damage induces stabilization and activation of the p53 transcription factor, which results in increased synthesis of the CDK inhibitory protein p21Cip1 (Fei and El-Deiry, 2003). Indeed, we observed enhanced Cdc6. destruction following DNA damage in a p53 and p21Cip1-dependent manner (Duursma and Agami, 2005). Interestingly, we also revealed that Cdc6 is regulated in a p53dependent manner in non-stressed cells. We demonstrate that siRNA mediated reduction of p53 protein levels results in more replicating cells, an effect that can be reversed by reducing Cdc6 protein level. Controlling the stability and activity of the licensing factors Both Cdc6 and Cdt1 are the licensing factors of DNA replication. Therefore, this key step in initiation of DNA replication appears to be regulated by several independent pathways. Whereas the protein abundance of Cdc6 in G1/S phase is positively regulated by cyclin E/CDK2 phosphorylation through protecting it from APCCdh1-dependent degradation, Cdt1 is negatively regulated during S-phase by Cyclin A phosphorylation in a SCFSkp2dependent manner (Li et al., 2003; Liu et al., 2004; Sugimoto et al., 2004). However, this. p53 E2F. APC. Protein degradation. G e m in in. C DK2/ C y c lin E. Transcription activation. Protein modification C dc6. S e r5 4 -P C dc6. G e m in in. C d t1. C d t1 ORC. S e r5 4 -P C dc6. C d t1 ORC. ORC. 1. Figure 1: A schematic model of the regulation of DNA replication licensing in G1 phase of the cell cycle. Licensing of replication origins occurs in a time-frame where geminin is degraded by the APC, resulting in active Cdt1, and Cdc6 is protected from APC-dependent degradation by CDK2/ cyclin E phosphorylation of serine 54.. 49.

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