Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere sequestration. Saccharomyces cerevisiae

The budding yeast telomere binding protein Rif1 (Rap1-interacting factor 1) plays an evolutionarily conserved role in the control of DNA replication timing, which operates through an interaction with the PP1 phosphatase. Rif1-PP1 has been proposed to inhibit origin firing by reversing the phosphorylation of key targets involved in replication initiation. However, it is not yet known if Rif1 binds directly to the replication origins that it controls. Here we show that in unperturbed yeast cells Rif1 primarily regulates late-replicating telomere-proximal origins. Using Chromatin Endogenous Cleavage (ChEC)-seq, we find that Rif1 is robustly detected at many late-replicating origins that we identify as targets of its inhibitory action. Abrogation of Rif1 telomere binding, through mutation of its Rap1 binding module, leads to increased Rif1 binding and late origin inhibition elsewhere in the genome. Our results support a model whereby Rif1 inhibits replication initiation by binding directly at origins, most of which are near telomeres, where Rif1 is concentrated through its interaction with telomere-bound Rap1 protein. Overall design: DNA replication timing was studied using leading strand polymerase Chromatin Immunoprecipitation seq (ChIP-seq) in cells released from an alpha factor block (G1 block) at different timepoints and quantitative sequencing of asynchronous FACS sorted S-phase cells (sort-seq). Protein binding of Rif1 was evaluated by Chromatin endonuclease cleavage seq (ChEC-seq)