Regulation of the DNA licensing protein Cdt1 in Xenopus laevis embryos

Abstract

During each cell cycle the DNA must be replicated accurately in order to maintain genomic integrity. To ensure faithful replication of the entire genome, DNA replication must be tightly controlled. This control is achieved through the process of DNA licensing in which pre-replicative complexes are assembled to prime the DNA for replication in the coming S-phase. To prevent re-licensing and subsequent re-replication, which would lead to genomic instability, DNA licensing must also be tightly controlled. The main mechanism of regulation of DNA licensing is through regulation of Cdt1 activity, a key component of DNA licensing. During the metazoan somatic cell cycle Cdt1 is regulated by proteolysis and inhibition by geminin. However there is evidence that the mechanisms of Cdt1 regulation during the short, rapid cleavage cell cycles of the early pre-MBT Xenopus embryo may differ. The results presented here show that upon expression of a deregulated, truncated version of Cdt1 in pre-MBT Xenopus embryos the cell cycle arrests with damaged DNA and evidence of checkpoint activation. This demonstrates that correct Cdt1 regulation is crucial for proper DNA licensing and pre-MBT embryonic cell cycle progression. There was no evidence of ubiquitination, degradation or phosphorylation of endogenous Cdt1. This suggests that changing interactions with geminin rather than proteolysis or post-translational modification provides the main mechanism of Cdt1 regulation in pre-MBT Xenopus embryos. The highly regulated N-terminal region of Cdt1 is capable of binding to DNA and the licensing component Orc1. This suggests that domains for DNA and Orc1 binding are also located at this region of the Cdt1 protein. However, a truncated Cdt1 construct lacking the N-terminal domain is still capable of licensing the DNA. Since the regulation of Cdt1 is crucial for correct DNA licensing, these interactions may therefore constitute redundant mechanisms to ensure the proper activity of Cdt1. Overall the results presented in this thesis show that in early Xenopus embryos Cdt1 regulation is crucial for faithful DNA licensing and cell cycle progression. In addition the main mechanism for regulation of Cdt1 is through dynamic interactions with geminin rather than post-translational modification or degradation during the pre-MBT embryonic cell cycle.