Rif1 controls DNA replication timing in yeast through the PP1 phosphatase Glc7.

The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK-dependent manner and that two proteins implicated in pre-RC activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK) phosphorylation in rif1 mutants. Rif1 also interacts with Dbf4 in yeast two-hybrid assays, further implicating this protein in direct modulation of pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK motif-dependent recruitment of Glc7 to telomeres and earlier replication of these regions in cells where the motifs are mutated. Our data thus link Rif1 to negative regulation of replication origin firing through recruitment of the Glc7 phosphatase.

[1]  K. Labib.  How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? , 2010, Genes & development.

[2]  A. Donaldson,et al.  Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast , 2011, The EMBO journal.

[3]  Bruce Stillman,et al.  Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. , 2006, Molecular cell.

[4]  J. Diffley,et al.  Regulating DNA replication in eukarya. , 2013, Cold Spring Harbor perspectives in biology.

[5]  David P. Toczyski,et al.  Damage-Induced Phosphorylation of Sld3 is Important to Block Late Origin Firing , 2010, Nature.

[6]  Zhaohui S. Qin,et al.  A Global Protein Kinase and Phosphatase Interaction Network in Yeast , 2010, Science.

[7]  E. Craig,et al.  Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. , 1996, Genetics.

[8]  Conrad A. Nieduszynski,et al.  Ku complex controls the replication time of DNA in telomere regions. , 2002, Genes & development.

[9]  M. Bollen,et al.  Docking motif-guided mapping of the interactome of protein phosphatase-1. , 2009, Chemistry & biology.

[10]  Yun Wah Lam,et al.  Displacement affinity chromatography of protein phosphatase one (PP1) complexes , 2008, BMC Biochemistry.

[11]  Anoushka Davé,et al.  Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity , 2014, Cell reports.

[12]  Vishnu Dileep,et al.  Mouse Rif1 is a key regulator of the replication‐timing programme in mammalian cells , 2012, The EMBO journal.

[13]  Hiroyuki Araki,et al.  CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast , 2007, Nature.

[14]  Nicolas H. Thomä,et al.  Rif1 and Rif2 Shape Telomere Function and Architecture through Multivalent Rap1 Interactions , 2013, Cell.

[15]  M. Raghuraman,et al.  The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation , 2011, Molecular biology of the cell.

[16]  J. Diffley,et al.  Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast , 2007, Nature.

[17]  Rakesh K Mishra,et al.  Functional diversification of yeast telomere associated protein, Rif1, in higher eukaryotes , 2011, BMC Genomics.

[18]  S. Bell,et al.  CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6. , 2011, Genes & development.

[19]  J. Diffley,et al.  The initiation of chromosomal DNA replication in eukaryotes. , 1990, Trends in genetics : TIG.

[20]  S. Bell,et al.  Eukaryotic Origin-Dependent DNA Replication In Vitro Reveals Sequential Action of DDK and S-CDK Kinases , 2011, Cell.

[21]  D. Shore,et al.  Delivery of yeast telomerase to a DNA break depends on the recruitment functions of Cdc13 and Est1. , 2004, Molecular cell.

[22]  D. Gottschling,et al.  Telomeric chromatin modulates replication timing near chromosome ends. , 1999, Genes & development.

[23]  J. Cannon.  Function of protein phosphatase-1, Glc7, in Saccharomyces cerevisiae. , 2010, Advances in applied microbiology.

[24]  Ryuichiro Nakato,et al.  Origin Association of Sld3, Sld7, and Cdc45 Proteins Is a Key Step for Determination of Origin-Firing Timing , 2011, Current Biology.

[25]  J. Diffley,et al.  Checkpoint Dependent Inhibition of DNA Replication Initiation by Sld3 and Dbf4 Phosphorylation , 2010, Nature.

[26]  John Bechhoefer,et al.  Reconciling stochastic origin firing with defined replication timing , 2009, Chromosome Research.

[27]  A. Donaldson.  Shaping time: chromatin structure and the DNA replication programme. , 2005, Trends in genetics : TIG.

[28]  Katsuhiko Shirahige,et al.  Rif1 is a global regulator of timing of replication origin firing in fission yeast. , 2012, Genes & development.

[29]  O. Aparicio,et al.  Location, location, location: it's all in the timing for replication origins. , 2013, Genes & development.

[30]  Fred Winston,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1990 .

[31]  H. Araki.  Initiation of chromosomal DNA replication in eukaryotic cells; contribution of yeast genetics to the elucidation. , 2011, Genes & genetic systems.

[32]  D. Shore,et al.  Yeast Ku protein plays a direct role in telomeric silencing and counteracts inhibition by Rif proteins , 1999, Current Biology.

[33]  Hisao Masai,et al.  Rif1 regulates the replication timing domains on the human genome , 2012, The EMBO journal.

[34]  M. Raghuraman,et al.  Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex , 2014, Genes & development.

[35]  D. Shore,et al.  A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation. , 1992, Genes & development.

[36]  Robbie Loewith,et al.  Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling. , 2009, Molecular cell.

[37]  D. Shore,et al.  Early Replication of Short Telomeres in Budding Yeast , 2007, Cell.