Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

Abstract

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.

Mechanisms and regulation of DNA replication initiation in eukaryotes

“…Although budding yeast ORC remains stably bound throughout the cell cycle (Diffley et al, 1994, Liang and Stillman, 1997, Fujita et al, 1998), vertebrate Orc1 is the target of CDK-dependent and CDK-independent mechanisms that remove it from chromatin (Figure 8) (Rowles et al, 1999, Findeisen et al, 1999, Natale et al, 2000, Li and DePamphilis, 2002, Li et al, 2004, Kreitz et al, 2001, Sun et al, 2002). In both yeast and metazoans, initiation factors are targeted for proteolysis in a CDK-dependent fashion, with budding yeast exclusively targeting Cdc6 (Piatti et al, 1995, Drury et al, 1997, Perkins et al, 2001, Mimura et al, 2004) and metazoans targeting both Orc1 and Cdc6 (Mendez et al, 2002, Tatsumi et al, 2003, Ohta et al, 2003, Lidonnici et al, 2004, Kalfalah et al, 2015). In S. pombe , Orc2 is also targeted by CDK to prevent re-licensing, but the mechanism utilized in this instance is unclear (Vas et al, 2001, Wuarin et al, 2002).…”

Early S-phase cell hypersensitivity to heat stress

“…27,28 Moreover, Cdc6 and geminin have been reported to partially reside in the centrosomes in interphase cells, which suggests the existence of previously unnoticed centrosome-related functions of Cdc6 and geminin. [28][29][30] To investigate the bases of HSinduced DNA re-replication and centrosome overduplication, we measured the levels of the geminin, Cdt1 and Cdc6 mRNAs in the early S-phase HeLa cells that had been heat-stressed (45 C, 30 min) and recovered at 37 C for different time intervals (0, 3, 6 and 24 hr). The control cells were not heat-stressed but were otherwise subjected to the same manipulations.…”

DNA replication licensing factor Cdc6 and Plk4 kinase antagonistically regulate centrosome duplication via Sas-6

“…After the initiation of DNA replication, Cdc6 is removed from the replication origin and translocated to the cytoplasm during S phase in a CDK2-phosphorylation-dependent manner35. Previous reports show that Cdc6 is detected on centrosomes during S and G2 phases3637. However, the function of Cdc6 in centrosome dynamics remains unknown.…”