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

Shin-ichiro Hiraga, Chandre Monerawela, Yuki Katou, Sophie Shaw, Kate R. M. Clark, Katsuhiko Shirahige, Anne D. Donaldson

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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.
Original languageEnglish
Article numbere46222
Pages (from-to)1-14
Number of pages14
JournalEMBO reports
Volume19
Issue number9
Early online date13 Aug 2018
DOIs
Publication statusPublished - 1 Sep 2018

Fingerprint

Saccharomycetales
Replication Origin
Hydroxyurea
Yeast
DNA Replication
DNA
Fungal Proteins
Centromere
Chromatin Immunoprecipitation
Telomere
G1 Phase
S Phase
Chromosomes
Yeasts
Binding Sites
Labeling
Chromatin
Genes

Keywords

  • Rif1
  • DNA replication origin
  • ChIP-Seq
  • Nascent DNA
  • Centromere

Cite this

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks. / Hiraga, Shin-ichiro; Monerawela, Chandre; Katou, Yuki; Shaw, Sophie; Clark, Kate R. M.; Shirahige, Katsuhiko; Donaldson, Anne D.

In: EMBO reports, Vol. 19, No. 9, e46222, 01.09.2018, p. 1-14.

Research output: Contribution to journalArticle

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note = "We thank Javier Garzon and Vamsi Krishna Gali for discussion and advice on methods, and Takashi Kubota for helpful comments on the manuscript. This work was supported by Cancer Research UK Programme Award A19059 to ADD and SH. KS was supported by Grant‐in‐Aid for Scientific Research on Priority Areas (15H05970 and 15K21761) from Ministry of Education, Culture, Sports, Science and Technology, Japan. Funding Cancer Research UK (CRUK) A19059 Ministry of Education, Culture, Sports, Science and Technology (MEXT) 15H0597015K21761 Data availability ChIP‐Seq data and corresponding input data were submitted to ArrayExpress under accession number E‐MTAB‐6736.",
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N1 - We thank Javier Garzon and Vamsi Krishna Gali for discussion and advice on methods, and Takashi Kubota for helpful comments on the manuscript. This work was supported by Cancer Research UK Programme Award A19059 to ADD and SH. KS was supported by Grant‐in‐Aid for Scientific Research on Priority Areas (15H05970 and 15K21761) from Ministry of Education, Culture, Sports, Science and Technology, Japan. Funding Cancer Research UK (CRUK) A19059 Ministry of Education, Culture, Sports, Science and Technology (MEXT) 15H0597015K21761 Data availability ChIP‐Seq data and corresponding input data were submitted to ArrayExpress under accession number E‐MTAB‐6736.

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