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

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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Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels

The Journal of Cell Biology ◽

10.1083/jcb.200911037 ◽

2010 ◽

Vol 188 (2) ◽

pp. 209-221 ◽

Cited By ~ 42

Author(s):

Alexander M. Thomson ◽

Peter J. Gillespie ◽

J. Julian Blow

Keyword(s):

Replication Timing ◽

S Phase ◽

Replication Initiation ◽

Cyclin Dependent Kinase ◽

Chromosomal Dna ◽

Replication Rate ◽

Checkpoint Kinases ◽

Replicative Stress ◽

Replication Factory ◽

Egg Extracts

In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase.

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

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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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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High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

10.1101/2021.05.14.443897 ◽

2021 ◽

Author(s):

Dashiell J Massey ◽

Amnon Koren

Keyword(s):

Dna Replication ◽

Replication Origin ◽

Replication Timing ◽

S Phase ◽

Human Cells ◽

Replication Initiation ◽

High Throughput Analysis ◽

Timing Variability ◽

Fixed Set ◽

Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

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X chromosome choice occurs independently of asynchronous replication timing

The Journal of Cell Biology ◽

10.1083/jcb.200405117 ◽

2005 ◽

Vol 168 (3) ◽

pp. 365-373 ◽

Cited By ~ 29

Author(s):

Joost Gribnau ◽

Sandra Luikenhuis ◽

Konrad Hochedlinger ◽

Kim Monkhorst ◽

Rudolf Jaenisch

Keyword(s):

X Chromosome ◽

Molecular Mechanisms ◽

Replication Timing ◽

S Phase ◽

X Inactivation ◽

Wild Type ◽

Gene Deletions ◽

Asynchronous Replication ◽

Skewed X Inactivation

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.

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Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly

The Journal of Cell Biology ◽

10.1083/jcb.200710019 ◽

2008 ◽

Vol 181 (2) ◽

pp. 241-254 ◽

Cited By ~ 101

Author(s):

Michael J. Emanuele ◽

Weijie Lan ◽

Miri Jwa ◽

Stephanie A. Miller ◽

Clarence S.M. Chan ◽

...

Keyword(s):

Cell Cycle ◽

Protein Phosphatase ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Aurora B Kinase ◽

Culture Cells ◽

Kinetochore Assembly ◽

M Phase ◽

Egg Extracts ◽

Cycle Dependent

The outer kinetochore binds microtubules to control chromosome movement. Outer kinetochore assembly is restricted to mitosis, whereas the inner kinetochore remains tethered to centromeres throughout the cell cycle. The cues that regulate this transient assembly are unknown. We find that inhibition of Aurora B kinase significantly reduces outer kinetochore assembly in Xenopus laevis and human tissue culture cells, frog egg extracts, and budding yeast. In X. leavis M phase extracts, preassembled kinetochores disassemble after inhibiting Aurora B activity with either drugs or antibodies. Kinetochore disassembly, induced by Aurora B inhibition, is rescued by restraining protein phosphatase 1 (PP1) activity. PP1 is necessary for kinetochores to disassemble at the exit from M phase, and purified enzyme is sufficient to cause disassembly on isolated mitotic nuclei. These data demonstrate that Aurora B activity is required for kinetochore maintenance and that PP1 is necessary and sufficient to disassemble kinetochores. We suggest that Aurora B and PP1 coordinate cell cycle–dependent changes in kinetochore assembly though phosphorylation of kinetochore substrates.

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