Human Cdt1 lacking the evolutionarily conserved region that interacts with MCM2-7 is capable of inducing re-replication.
Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.
Teer
Jamie K
JK
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts 02115, USA.
Dutta
Anindya
A
D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism; D017124:Conserved Sequence; D004261:DNA Replication; D019143:Evolution, Molecular; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D008954:Models, Biological; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016196:S Phase
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2