ABSTRACT
The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.
Subject(s)
Cell Cycle Proteins/genetics , Chromatin/chemistry , DNA Replication , DNA-Binding Proteins/genetics , Geminin/genetics , Ovum/chemistry , Xenopus Proteins/genetics , Xenopus laevis/genetics , Animals , Binding Sites , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromatin/metabolism , Cloning, Molecular , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Female , Geminin/metabolism , Gene Expression , Male , Mutation , Ovum/cytology , Ovum/metabolism , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spermatozoa/chemistry , Spermatozoa/cytology , Spermatozoa/metabolism , Structure-Activity Relationship , Xenopus Proteins/metabolism , Xenopus laevis/metabolismABSTRACT
Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.
