| Successful progression through S phase of the cell cycle requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion. In this dissertation, I studied the regulation of replication initiators which represents the first step in the replication process and hence subject to multiple control mechanisms. Although the requirement of cyclin dependent kinases (CDKs) for DNA replication is well established, the mechanism by which substrates are recruited to CDKs was unknown. In the first aim of this dissertation we examined the role of a peptide sequence referred to as the cyclin binding motif (Cy motif) in substrate phosphorylation by CDKs. Using a kinetic analysis we demonstrated that the Cy motif and phosphoacceptor site constitute a bipartite recognition motif for CDKs suggesting that the Cy motif may be responsible for the different substrate specificities of cyclin/CDK complexes. The second aim of this dissertation was to determine the importance of CDK phosphorylation on the regulation of the replication initiator Cdt1 by the F-box protein Skp2. We discovered that the N-terminus of Cdt1 is required for its degradation during S phase independent of Skp2 and cyclin binding. However, Skp2 and CDK phosphorylation is important for inhibiting re-replication caused by Cdt1 overexpression. Inhibiting Cdt1 degradation resulted in a significantly delayed entry into and completion of S phase demonstrating the importance of properly regulating Cdt1.; Although we have identified numerous proteins that function at origins to initiate replication, we are still limited in our knowledge of what specifies a replication origin in vivo. Therefore in the third aim of this dissertation, we developed an artificial origin in vivo by recruiting replication factors to a specific DNA sequence. Different components of the ORC complex as well as Cdc6 stimulated replication when recruited to DNA using the GAL4 operon system. The artificial origin recapitulates many of the regulatory features of physiological origins and should be valuable for studies on replication initiation in mammalian cells. |
