Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

Abstract

We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G 1 , but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin͞nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.In all eukaryotic cells, DNA replication is a tightly regulated process that is strictly coupled to the progression of the cell cycle. It occurs only during S phase, and initiation of DNA replication occurs at discrete chromosomal locations (replication origins). When DNA replication is initiated, the cell must ensure that all its genome is replicated and that the replication of every DNA section occurs once and only once during the cell cycle. In the budding yeast, Cdc6p plays a unique role in regulating DNA replication. It is essential for initiation of DNA replication and required for assembly and maintenance of the prereplication complexes (pre-RCs) at replication origins (1, 2). In contrast to the origin recognition complex (ORC) and minichromosome maintenance (Mcm) proteins, Cdc6p is expressed only in G 1 phase of the Saccharomyces cerevisiae cell cycle (3, 4). Cdc6p and its Schizosaccharomyces pombe homologue Cdc18 physically interact with ORC, and Cdc6p is required for the loading of Mcm proteins at the replication origins in G 1 (5, 6). Cdc6p is a nucleotidedependent loading factor related to the eukaryotic and prokaryotic clamp loaders, such as PCNA (7,8). Both Cdc6p and Cdc18 also are associated with Cdks in vivo and can be phosphorylated by Cdks in vitro (9, 10). It is thought that Cdk phosphorylation of Cdc6p and Cdc18 triggers their degradation through a ubiquitination-mediated protein-degradation pathway (11-13). A gain-of-function Cdc6p mutant displays promiscuous initiation of DNA replication and promotes constant Mcm proteins association with chromatin throughout the cell cycle (14). Overexpression of a Cdc18 mutant that cannot be phosphorylated by Cdks in vitro causes greater DNA overreplication in Schizosaccharomyces pombe than expression of wild-type Cdc18 (13, 15). Taken together, these findings indicate that Cdc6p͞C...

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

“…9 Also in our hands exclusive cytosolic localization of Cdc6-YFP during late stages of the cell cycle could as well be reverted into a more nuclear localization when cells were treated with the specific Crm1 inhibitor Leptomycin B (Fig. 2D).…”

“…Since this takes place several hours after stimulation, it was concluded that Cdc6 stabilization takes place during S phase. [9][10][11]18 We wanted to test whether this regulation applies to YFP-tagged Cdc6, too. When untransfected HT-1080 cells (Fig.…”

“…15,16 Phosphorylation also regulates the cellular localization of Cdc6. It has been demonstrated that phosphorylation of Cdc6 by Cyclin A/Cdk2 translocates the protein to the cytoplasm during S phase, 9,10,[17][18][19] and some authors suggested that Cdc6 might first be exported to the cytoplasm, and then degraded in an APC-dependent manner. 20,21 This interpretation is however challenged by reports demonstrating that at least a fraction of Cdc6 protein remains nuclear and chromatin-bound even in Sphase.…”

“…It is well established that the stability of Cdc6 protein is regulated by phosphorylation at 3 canonical CDK sites within its N-terminal domain, 8,9 which depends on acetylation by the acetyltransferase GCN5 at specific residues nearby. 10 In quiescent mammalian cells the anaphase promoting complex (APC) E3 ubiquitin ligase provides for constant proteasomal degradation of Cdc6.…”

DNA replication licensing and cell cycle kinetics of oligodendroglial tumours

“…We have examined expression of the standard proliferation marker Ki67, the DNA replicationlicensing factor Mcm2 and the repressor of origin-licensing Geminin during the proliferative cell cycle, using a novel, nonchemical method for cell synchronisation (membrane elution; Thornton et al, 2002;Helmstetter et al, 2003; Figure 1C). We have employed membrane elution because use of chemical methods for cell synchronisation may account in part for reported discrepancies in temporal periodicities and subcellular localisation of replication proteins such as Orc1 (Pak et al, 1997;Kreitz et al, 2001;Okuno et al, 2001;Li and DePamphilis, 2002) and Cdc6 (Fujita et al, 1999;Jiang et al, 1999;Mendez and Stillman, 2000;Petersen et al, 2000).…”