Rif1 Controls DNA Replication Timing in Yeast through the PP1 Phosphatase Glc7

•Rif1 interacts through RVxF/SILK motifs with yeast PP1 phosphatase Glc7•Rif1-Glc7 inhibits the Dbf4-dependent kinase pathway for DNA replication initiation•Rif1-Glc7 regulates the phosphorylation of proteins involved in pre-RC activation•Rif1-Glc7 is recruited to telomeres and regulates telomeric replication timing

SummaryThe 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.

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