Regulation of the DNA licensing protein Cdt1 in Xenopus laevis Embryos.

In every organism, it is essential that DNA is replicated accurately only once per cell cycle in order to prevent potentially cancerous changes to the genome. Proper licensing of the DNA prior replication during S-phase ensures that the DNA is replicated faithfully with no sections left under- or over- replicated. DNA licensing is complete once the proteins of the pre-replication complex (pre-RC) are successfully loaded at the replication origins of the DNA. In somatic cells strict regulation of the pre-RC component Cdt1 ensures appropriate DNA licensing. In metazoan somatic cells a natural inhibitor of Cdt1, geminin, regulates Cdt1 activity along with post-translational modification and degradation of Cdt1. However the mechanisms of Cdt1 regulation in early embryonic cell cycles are unclear. To investigate Cdt1 regulation during the embryonic cell cycle, truncated Cdt1 constructs were designed: Cdt1_1-243 and Cdt1_243-620. The first one represented N terminus of Cdt1 known to be a regulatory part of the protein. The latter lacked the regulatory domain but included geminin and DNA binding sites. The constructs were expressed in Xenopus embryos and their effects on cell division and development was determined. Here we show that upon expression in Xenopus embryos, Cdt1_243-620 caused the affected cells to arrest leading to abnormal embryonic development. The cells arrest in a licensed state with evidence of DNA damage and checkpoint activation suggesting the occurrence of DNA re-replication. In agreement with this a further Cdt1 construct, designed to be defective at DNA licensing, did not result in cell cycle arrest when expressed in Xenopus embryos. Taken together this suggests that Cdt1_243-620 causes re-licensing and re-replication leading to DNA damage which then triggers the cell cycle arrest observed. Furthermore, we show that, in Xenopus egg extract the N-terminal region of Cdt1, Cdt1_1-243 undergoes degradation under conditions of high DNA levels. Cdt1_1-243 is also capable of crossing the nuclear membrane and binding DNA in both Xenopus embryos and the in the in vitro Xenopus egg extract system. In conclusion these results show that correct Cdt1 regulation is essential to maintain cell cycle integrity and cell division during embryonic development. In addition, the N-terminal region of the Cdt1 protein is essential for its regulation and ensuring correct embryonic cell division and development.