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

Vamsi Krishna Gali; Chandre Monerawela; Yassine Laksir; Shin-Ichiro Hiraga; Anne D Donaldson

doi:10.1371/journal.pgen.1011044

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

Vamsi Krishna Gali, Chandre Monerawela, Yassine Laksir, Shin-Ichiro Hiraga, Anne D Donaldson^* (Corresponding Author)

^*Corresponding author for this work

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

Abstract

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.

Original language	English
Article number	e1011044
Number of pages	24
Journal	PLoS Genetics
Volume	19
Issue number	11
Early online date	13 Nov 2023
DOIs	https://doi.org/10.1371/journal.pgen.1011044
Publication status	Published - 1 Dec 2023

Bibliographical note

Funding: This work was supported by Cancer Research UK Programme Award DRCPGM\100013 (to ADD and SH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

Data Availability: ChIP-Seq data is uploaded to ArrayExpress under accession number E-MTAB-13451 (Rif1-9V5 datasets), and E-MTAB-13452 (Rif1-13Myc datasets).

Keywords

DNA replication
Phosphorylation
Saccharomyces cerevisiae
Yeast
genomics
auxins
Nucleases
helicases

UN SDGs

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

Access to Document

10.1371/journal.pgen.1011044Licence: CC BY

Gali_etal_PG_Checkpoint_Phosphorylation_Sites_VoR
© 2023 Gali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. https://creativecommons.org/licenses/by/4.0/
Final published version, 3.3 MBLicence: CC BY

ChIP-Seq analysis of V5-tagged budding yeast Rif1
Hiraga, S. (Creator), EMBL-EBI, 19 Nov 2023
https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-13452
Dataset
ChIP-Seq analysis of Myc-tagged budding yeast Rif1
Hiraga, S. (Creator), EMBL-EBI, 19 Nov 2023
https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-13451
Dataset

Cite this

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

abstract = "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.",

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AU - Laksir, Yassine

AU - Hiraga, Shin-Ichiro

AU - Donaldson, Anne D

N1 - Funding: This work was supported by Cancer Research UK Programme Award DRCPGM\100013 (to ADD and SH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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N2 - 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.

AB - 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.

KW - DNA replication

KW - Phosphorylation

KW - Saccharomyces cerevisiae

KW - Yeast

KW - genomics

KW - auxins

KW - Nucleases

KW - helicases

U2 - 10.1371/journal.pgen.1011044

DO - 10.1371/journal.pgen.1011044

M3 - Article

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JO - PLoS Genetics

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ER -

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

Abstract

Bibliographical note

Data Availability Statement

Keywords

UN SDGs

Access to Document

Fingerprint

Datasets

ChIP-Seq analysis of V5-tagged budding yeast Rif1

ChIP-Seq analysis of Myc-tagged budding yeast Rif1

Cite this