Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

Publication date: 7 March 2017
Source:Cell Reports, Volume 18, Issue 10
Author(s): Robert C. Alver, Gaganmeet Singh Chadha, Peter J. Gillespie, J. Julian Blow
Dbf4-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.

Graphical abstract

Teaser

Alver et al. find that DDK-dependent MCM2-7 phosphorylation is opposed by Rif1-targeted PP1. Lack of Rif1 increases MCM phosphorylation and abolishes the ability to block origin firing, even when undergoing replication stress. In the absence of CHK1, the stability of stalled replisomes is severely compromised upon Rif-PP1-mediated dephosphorylation.


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