title: Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.
abstract: 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.
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Alver
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Gaganmeet Singh
authors.initials: GS
authors.lastname: Chadha
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Gillespie
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. Electronic address: j.j.blow@dundee.ac.uk.
authors.forename: J Julian
authors.initials: JJ
authors.lastname: Blow
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1 / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics; D016196:S Phase; D034501:Telomere-Binding Proteins / Q000378:metabolism*; D014982:Xenopus laevis; D000099272:Replisomes
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; D004259:DNA-Directed DNA Polymerase; D064110:Minichromosome Maintenance Proteins; D009097:Multienzyme Complexes; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D034501:Telomere-Binding Proteins; D000099272:Replisomes; C492743:Rif1 protein, human; C576597:ATR protein, human; C000605867:CHEK1 protein, human
keywords: Cdc7; DNA damage; DNA replication; MCM; Rif1; Xenopus egg cell-free system; checkpoint signaling; human tissue culture