Abstract
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.
Highlights
Chromosomes are highly dynamic, and chromatin changes its structural composition during functional processes and at different cell cycle stages
To obtain a better understanding of Rif1 function in DNA replication and genome maintenance, we examined the chromatin association patterns of Rif1 by next-generation sequencing analysis of chromatin immunoprecipitation (ChIP) samples (ChIP-Seq), which provides an improved dynamic range of analysis compared to microarrays
Structural studies revealed two domains within Rif1 that interact with Rap1: the Rap1binding motif (RBM) and a C-terminal domain (CTD; Fig 1A) [30]
Summary
Chromosomes are highly dynamic, and chromatin changes its structural composition during functional processes and at different cell cycle stages. Rif was originally identified in the budding yeast Saccharomyces cerevisiae as a component of telomeric chromatin that regulates telomere length [1,2]. Rap recognises a TG-rich motif present upstream of genes it regulates This recognition motif occurs within the telomeric terminal TG repeat sequences, and multiple copies of Rap bind telomeres [4]. While originally isolated for its role in binding Rap at telomeres, Rif (Rap1-Interacting Factor 1) has recently been identified as an important regulator of DNA replication initiation, in a function conserved from yeast to human [5,6,7,8,9,10,11]. Rif ChIP peaks are a mixture of narrow peaks (e.g. peaks at ARS elements in G1 phase) and broad peaks (e.g. ORF peaks and peaks at early origins in HU arrest). Positions of centromeres were obtained from 20110203 release of SGD
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