Checkpoint phosphorylation sites on budding yeast Rif1 protect nascent DNA from degradation by Sgs1-Dna2

In budding yeast the Rif1 protein is important for protecting nascent DNA at blocked replication forks, but the mechanism has been unclear. Here we show that budding yeast Rif1 must interact with Protein Phosphatase 1 to protect nascent DNA. In the absence of Rif1, removal of either Dna2 or Sgs1 prevents nascent DNA degradation, implying that Rif1 protects nascent DNA by targeting Protein Phosphatase 1 to oppose degradation by the Sgs1-Dna2 nuclease-helicase complex. This functional role for Rif1 is conserved from yeast to human cells. Yeast Rif1 was previously identified as a target of phosphorylation by the Tel1/Mec1 checkpoint kinases, but the importance of this phosphorylation has been unclear. We find that nascent DNA protection depends on a cluster of Tel1/Mec1 consensus phosphorylation sites in the Rif1 protein sequence, indicating that the intra-S phase checkpoint acts to protect nascent DNA through Rif1 phosphorylation. Our observations uncover the pathway by which budding yeast Rif1 stabilises newly synthesised DNA, highlighting the crucial role Rif1 plays in maintaining genome stability from lower eukaryotes to humans. Author summaryGenome instability is a leading factor contributing to cancer. Maintaining efficient error-free replication of the genome is key to preventing genome instability. During DNA replication, replication forks can be stalled by external and intrinsic obstacles, leading to processing of nascent DNA ends to enable replication restart. However, the nascent DNA must be protected from excessive processing to prevent terminal fork arrest, which could potentially lead to more serious consequences including failure to replicate some genome sequences. Using a nascent DNA protection assay we have investigated the role of the budding yeast Rif1 protein at blocked replication forks. We find that Rif1 protects nascent DNA through a mechanism that appears conserved from yeast to humans. We show that budding yeast Rif1 protects nascent DNA by targeting Protein Phosphatase 1 activity to prevent degradation of nascent DNA by the Sgs1-Dna2 helicase-nuclease complex. Furthermore, we find that Rif1 phosphorylation by the checkpoint pathway during replication stress is crucial for this function. Our results indicate that the S phase checkpoint machinery acts by phosphorylating Rif1 to protect nascent DNA, providing important clues concerning the conserved role of Rif1 in regulating events when replication is challenged.