Control of DNA Replication by the Cdt1/Geminin ComplexPublic Deposited
It is critical to genomic integrity that DNA is replicated completely and faithfully during each cell cycle. The essential replication factor Cdt1 is a critical protein in preventing reinitiation of replication. Overexpression of Cdt1 causes re-replication in p53 null cells and transforms NIH3T3 cells, causing them to form tumors in nude mice. We sought to determine the mechanisms that shut off Cdt1 activity and prevent re-replication. We developed an in vitro system using replication extract from Xenopus laevis eggs to analyze the mechanisms that regulate Cdt1. We found that the C-terminus of Cdt1 is required for replication whereas the N-terminus is dispensable, suggesting this region plays a regulatory role. We mapped the degradation signal and binding site for the replication inhibitor Geminin to the N-terminus. We found that both ubiquitin-dependent degradation and binding to Geminin shut off Cdt1 and prevent re-replication. Our data suggests that Geminin is required to prevent re-replication specifically during G2 phase. We found that Cdt1 is phosphorylated on 15 sites during metaphase. Cdt1 with mutations at ten putative CDK phosphorylation sites shows variable activity, suggesting that phosphorylation may consist of both activating and inhibitory mechanisms. We also tested the activity of Cdt1 mutants in vivo using Xenopus as a model organism. Geminin deficient Xenopus embryos arrest in G2 phase of the cell cycle because of activation of the replication checkpoint. We show here that expression of a Cdt1 mutant that does not bind Geminin or a mutant that is not degraded reproduces the phenotype of Geminin deficiency. This suggests that these mechanisms shut off Cdt1 in vivo and that the cell cycle arrest seen in Geminin deficient Xenopus embryos is caused by re-replication. Geminin interacts with a number of transcription factors and chromatin remodeling proteins in ways that suggest it may inhibit differentiation when cells are proliferating during development. We show that Geminin deficient Xenopus embryos fail to express the early embryonic genes Brachyury and Goosecoid. This gene expression defect is reproduced by expressing mis-regulated Cdt1, suggesting it is a secondary effect of the cell cycle arrest caused by re-replication.