Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks. Synopsis This study identifies genome‐wide Rif1 bindings sites in budding yeast. Rif1 binds both replication origins and stalled replication forks and protects nascent DNA from degradation. ChIP‐Seq analysis revealed Rap1‐dependent and –independent budding yeast Rif1 chromosomal binding sites. Rif1 binds early and late origins without apparent preference. Rif1 binds stalled replication forks or post‐replicative chromatin under replication stress, and protects nascent DNA from degradation. Rif1 associates with centromeres in S phase. Graphical Abstract This study identifies genome‐wide Rif1 bindings sites in budding yeast. Rif1 binds both replication origins and stalled replication forks and protects nascent DNA from degradation.