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

Mapping Intimacies ◽

10.1101/2020.06.25.170571 ◽

2020 ◽

Author(s):

Chandre Monerawela ◽

Shin-ichiro Hiraga ◽

Anne D. Donaldson

Keyword(s):

Protein Phosphatase ◽

Genome Instability ◽

Budding Yeast ◽

S Phase ◽

Protein Phosphatase 1 ◽

Phosphorylation Sites ◽

Replication Forks ◽

Dna Protection ◽

S Phase Checkpoint

AbstractIn 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.Author summaryGenome instability is a leading factor contributing to cancer. Maintaining efficient error-free replication of the genome is key to preventing genome instability. During DNA replication, replication forks can be stalled by external and intrinsic obstacles, leading to processing of nascent DNA ends to enable replication restart. However, the nascent DNA must be protected from excessive processing to prevent terminal fork arrest, which could potentially lead to more serious consequences including failure to replicate some genome sequences. Using a nascent DNA protection assay we have investigated the role of the budding yeast Rif1 protein at blocked replication forks. We find that Rif1 protects nascent DNA through a mechanism that appears conserved from yeast to humans. We show that budding yeast Rif1 protects nascent DNA by targeting Protein Phosphatase 1 activity to prevent degradation of nascent DNA by the Sgs1-Dna2 helicase-nuclease complex. Furthermore, we find that Rif1 phosphorylation by the checkpoint pathway during replication stress is crucial for this function. Our results indicate that the S phase checkpoint machinery acts by phosphorylating Rif1 to protect nascent DNA, providing important clues concerning the conserved role of Rif1 in regulating events when replication is challenged.

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Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1

Open Biology ◽

10.1098/rsob.130138 ◽

2014 ◽

Vol 4 (1) ◽

pp. 130138 ◽

Cited By ~ 18

Author(s):

Wei Theng Poh ◽

Gaganmeet Singh Chadha ◽

Peter J. Gillespie ◽

Philipp Kaldis ◽

J. Julian Blow

Keyword(s):

Protein Phosphatase ◽

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Protein Phosphatase 1 ◽

Checkpoint Kinases ◽

Anti Cancer ◽

Egg Extracts ◽

Mcm Proteins

The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.

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Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA

Molecular Biology of the Cell ◽

10.1091/mbc.e19-03-0156 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2771-2789

Author(s):

Jeff Julius ◽

Jie Peng ◽

Andrew McCulley ◽

Chris Caridi ◽

Remigiusz Arnak ◽

...

Keyword(s):

Mitotic Spindle ◽

Replication Fork ◽

Budding Yeast ◽

S Phase ◽

Force Balance ◽

Replication Forks ◽

Origin Firing ◽

Spindle Structure ◽

Kinetochore Function ◽

S Phase Checkpoint

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

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The Essential Role of Protein Phosphatase-1 in Mitogenic Signaling and Breast Cancer

10.21236/ada398153 ◽

2001 ◽

Author(s):

Carey Oliver ◽

Shirish Shenolikar

Keyword(s):

Breast Cancer ◽

Protein Phosphatase ◽

Essential Role ◽

Protein Phosphatase 1 ◽

Mitogenic Signaling

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Novel role of the protein phosphatase 1 regulatory subunit PPP1R3A in atrial fibrillation

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2018.07.077 ◽

2018 ◽

Vol 124 ◽

pp. 108

Author(s):

Katherina Alsina ◽

Mohit Hulsurkar ◽

Chunxia Yao ◽

Barbara Langer ◽

David Chiang ◽

...

Keyword(s):

Atrial Fibrillation ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Regulatory Subunit

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Ref2, a regulatory subunit of the yeast protein phosphatase 1, is a novel component of cation homoeostasis

Biochemical Journal ◽

10.1042/bj20091909 ◽

2010 ◽

Vol 426 (3) ◽

pp. 355-364 ◽

Cited By ~ 12

Author(s):

Jofre Ferrer-Dalmau ◽

Asier González ◽

Maria Platara ◽

Clara Navarrete ◽

José L. Martínez ◽

...

Keyword(s):

Protein Phosphatase ◽

Living Organism ◽

Calcium Sensitivity ◽

Growth Conditions ◽

Protein Phosphatase 1 ◽

Regulatory Subunit ◽

Alkaline Ph ◽

Yeast Protein ◽

Increased Sensitivity

Maintenance of cation homoeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homoeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (sodium, lithium and calcium) and alkaline pH. Among 18 candidates, only deletion of REF2 (RNA end formation 2) yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. The Ref2F374A mutation, which renders it unable to bind Glc7, did not rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homoeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na+-ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homoeostasis may be unrelated to its previously identified function in the formation of mRNA via the APT (for associated with Pta1) complex.

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MRX (Mre11/Rad50/Xrs2) Mutants Reveal Dual Intra-S-Phase Checkpoint Systems in Budding Yeast

Cell Cycle ◽

10.4161/cc.4.8.1854 ◽

2005 ◽

Vol 4 (8) ◽

pp. 4073-4077 ◽

Cited By ~ 1

Author(s):

Catherine A. Andrews ◽

Duncan J. Clarke

Keyword(s):

Budding Yeast ◽

S Phase ◽

S Phase Checkpoint

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Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

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Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase

Molecular and Cellular Biology ◽

10.1128/mcb.00260-17 ◽

2017 ◽

Vol 37 (22) ◽

Cited By ~ 2

Author(s):

Michael C. Reubens ◽

Sophie Rozenzhak ◽

Paul Russell

Keyword(s):

Genome Stability ◽

Genome Instability ◽

Replication Stress ◽

S Phase ◽

Dna Lesions ◽

Brct Domain ◽

Replication Forks ◽

Content Type ◽

Domain Protein ◽

Chromosome Integrity

ABSTRACT DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

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