Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication

Abstract

To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1a associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1a/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.

Dynamic regulation of histone H3K9 is linked to the switch between replication and transcription at the Dbf4 origin-promoter locus

“…[34][35][36][37] Interestingly, however, we have recently found that human Dbf4 protein levels are not dramatically changed during the cell cycle, with only a slight decrease in the Dbf4 protein level during a brief period after prometaphase. 38 Our data thus showed that the levels of Dbf4 transcription and proteins are not necessarily tightly coupled, raising the possibility that the cell cycle regulation of Dbf4 transcription activity may have an unknown function. 38 We previously identified a strong early-firing origin in the human Dbf4 gene promoter locus (Fig.…”

“…38 Our data thus showed that the levels of Dbf4 transcription and proteins are not necessarily tightly coupled, raising the possibility that the cell cycle regulation of Dbf4 transcription activity may have an unknown function. 38 We previously identified a strong early-firing origin in the human Dbf4 gene promoter locus (Fig. S1).…”

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks

“…As CDC7-dependent phosphorylation of MCM2 at S40 is cell cycle regulated and dependent on a priming kinase phosphorylating S41 36 , we also assessed CDC7 phosphorylation in cells arrested in mitosis. Under these conditions, CDC7 has been shown to be phosphorylated at multiple residues, some of which are potentially due to autophosphorylation, resulting in an electrophoretic mobility shift during SDS-PAGE 37,38 . MCF10A EditR, MCF10A DBF4- and DRF1-deficient cells were arrested in mitosis using nocodazole, an inhibitor of microtubule polymerisation, and then protein extracts were prepared and analysed by immunoblotting.…”

The Hippo Signaling Pathway in Cancer: A Cell Cycle Perspective

“…Besides, unlike the mammalian Hippo pathway effector YAP that becomes inactive upon cytoplasmic retention, the Schizosaccharomyces pombe Clp1 more closely resembles its Saccharomyces cerevisiae ortholog Cdc14 that exerts its functions in the cytosol. There is evidence that Cdc7 is phosphorylated by CDK1 in mitosis and dephosphorylated by PP1α as cells exit mitosis [ 99 ]. Thus, similar to its Saccharomyces cerevisiae peer Cdc15, Cdc7 is more likely to facilitate mitotic exit in a phosphorylation-free state, which is another difference from its mammalian peer MST1/2 that are activated by phosphorylation.…”