Abstract
SummaryDbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.
Highlights
The ordered assembly of replication proteins onto chromatin is highly regulated to ensure that DNA is precisely copied only once each cell cycle
We used Xenopus egg extracts to evaluate the hyper-phosphorylation of Mcm4 and the phosphorylation of Mcm2 at the Xenopus orthologous S40 and S53 residues (S25 and S38, respectively) as readouts for the dependent kinases (DDKs) activity required for DNA replication (Figure 1A)
Treatment with the DDK inhibitor PHA-767491 resulted in completely dephosphorylated Mcm4
Summary
The ordered assembly of replication proteins onto chromatin is highly regulated to ensure that DNA is precisely copied only once each cell cycle. The key target of DDK for the initiation of DNA replication is the Mcm complex. The contribution of each of these phosphorylation events to initiation is not well understood, a key consequence of the hyperphosphorylation of Mcm is the relief of an inhibitory signal that prevents replication (Sheu and Stillman, 2006, 2010). Cells lacking this Mcm N-terminal tail no longer require DDK activity to complete replication. In Xenopus laevis egg extracts, Mcm hyper-phosphorylation by DDK occurs primarily on chromatin (Poh et al, 2014), though experiments in mammalian cells have suggested that DDK phosphorylation of Mcm occurs primarily when it is not bound to DNA (Montagnoli et al, 2006)
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