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

doi:10.15252/embr.201846222

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^* (Corresponding Author)

^*Corresponding author for this work

The University of Tokyo

Research output: Contribution to journal › Article › peer-review

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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 language	English
Article number	e46222
Number of pages	16
Journal	EMBO reports
Volume	19
Issue number	9
Early online date	13 Aug 2018
DOIs	https://doi.org/10.15252/embr.201846222 https://doi.org/10.15252/embr.201948152
Publication status	Published - 1 Sept 2018

Bibliographical 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

Corrigendum to Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (EMBO reports, (2018), 19, 9, (e46222), 10.15252/embr.201846222)
It came to the authors’ attention that in the original paper, an incorrect conversion factor was used for the pixel to micrometre conversion when analysing microscope images of combed fibres. The conversion factor used was 7.2 pixels = 1 µm instead of 13.9 pixels = 1 µm.

Data Availability Statement

Data availability
ChIP‐Seq data and corresponding input data were submitted to ArrayExpress under accession number E‐MTAB‐6736.

Keywords

Rif1
DNA replication origin
ChIP-Seq
Nascent DNA
Centromere

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.15252/embr.201846222Licence: CC BY
10.15252/embr.201948152

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks
© 2018 The Authors. This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. https://creativecommons.org/licenses/by/4.0/
Final published version, 1.31 MBLicence: CC BY

Cite this

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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.",

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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 Corrigendum to Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks (EMBO reports, (2018), 19, 9, (e46222), 10.15252/embr.201846222) It came to the authors{\textquoteright} attention that in the original paper, an incorrect conversion factor was used for the pixel to micrometre conversion when analysing microscope images of combed fibres. The conversion factor used was 7.2 pixels = 1 µm instead of 13.9 pixels = 1 µm. ",

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AU - Donaldson, Anne D.

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

Abstract

Bibliographical note

Data Availability Statement

Keywords

UN SDGs

Access to Document

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Anne Donaldson

Centre for Genome-Enabled Biology and Medicine

Cite this

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

Abstract

Bibliographical note

Data Availability Statement

Keywords

UN SDGs

Access to Document

Other files and links

Fingerprint

Profiles

Anne Donaldson

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Centre for Genome-Enabled Biology and Medicine

Cite this