Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis
| Autor: | Christian Mielke, Elke Berg, Fritz Boege, Faiza Kalfalah, Morten O. Christensen, Wilhelm G. Dirks, René M. Linka |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
DNA Replication
licensing Blotting Western Green Fluorescent Proteins cell cycle control Mitosis Cell Cycle Proteins Eukaryotic DNA replication Pre-replication complex Genomic Instability DNA replication factor CDT1 G2 phase Bacterial Proteins Control of chromosome duplication Report Proliferating Cell Nuclear Antigen Humans Molecular Biology Centrosome biology Nuclear Proteins S-phase-promoting factor Cell Biology Flow Cytometry Immunohistochemistry Chromatin Cell biology Luminescent Proteins Licensing factor Microscopy Fluorescence biology.protein Origin recognition complex nuclear export Developmental Biology |
| Zdroj: | Cell Cycle |
| ISSN: | 1551-4005 1538-4101 |
| DOI: | 10.1080/15384101.2014.1000182 |
| Popis: | To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner. |
| Databáze: | OpenAIRE |
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