An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

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Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0020 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4374-4382 ◽

Cited By ~ 13

Author(s):

Ling Yin ◽

Alexandra Monica Locovei ◽

Gennaro D'Urso

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

S Phase ◽

Replication Initiation ◽

Dna Damage Checkpoint ◽

Origin Firing ◽

Dna Replication Initiation ◽

Fork Stalling ◽

S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

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Identification of the critical replication targets of CDK reveals direct regulation of replication initiation factors by the embryo polarity machinery in C. elegans

PLoS Genetics ◽

10.1371/journal.pgen.1008948 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1008948

Author(s):

Vincent Gaggioli ◽

Manuela R. Kieninger ◽

Anna Klucnika ◽

Richard Butler ◽

Philip Zegerman

Keyword(s):

Direct Interaction ◽

Early Embryo ◽

S Phase ◽

Replication Initiation ◽

Dna Replication Initiation ◽

C Elegans ◽

Initiation Factors ◽

Physiological Importance ◽

Essential Function ◽

Phase Length

During metazoan development, the cell cycle is remodelled to coordinate proliferation with differentiation. Developmental cues cause dramatic changes in the number and timing of replication initiation events, but the mechanisms and physiological importance of such changes are poorly understood. Cyclin-dependent kinases (CDKs) are important for regulating S-phase length in many metazoa, and here we show in the nematode Caenorhabditis elegans that an essential function of CDKs during early embryogenesis is to regulate the interactions between three replication initiation factors SLD-3, SLD-2 and MUS-101 (Dpb11/TopBP1). Mutations that bypass the requirement for CDKs to generate interactions between these factors is partly sufficient for viability in the absence of Cyclin E, demonstrating that this is a critical embryonic function of this Cyclin. Both SLD-2 and SLD-3 are asymmetrically localised in the early embryo and the levels of these proteins inversely correlate with S-phase length. We also show that SLD-2 asymmetry is determined by direct interaction with the polarity protein PKC-3. This study explains an essential function of CDKs for replication initiation in a metazoan and provides the first direct molecular mechanism through which polarization of the embryo is coordinated with DNA replication initiation factors.

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ERK Activity and G1 Phase Progression: Identifying Dispensable Versus Essential Activities and Primary Versus Secondary Targets

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0908 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1457-1463 ◽

Cited By ~ 42

Author(s):

Jessie Villanueva ◽

Yuval Yung ◽

Janice L. Walker ◽

Richard K. Assoian

Keyword(s):

Cyclin D1 ◽

Map Kinases ◽

S Phase ◽

G1 Phase ◽

Cyclin Dependent Kinases ◽

Mechanistic Analysis ◽

Phase Progression ◽

Erk Activity ◽

Phase Entry ◽

Stimulation Of

The ERK subfamily of MAP kinases is a critical regulator of S phase entry. ERK activity regulates the induction of cyclin D1, and a sustained ERK signal is thought to be required for this effect, at least in fibroblasts. We now show that early G1 phase ERK activity is dispensable for the induction of cyclin D1 and that the critical ERK signaling period is restricted to 3–6 h after mitogenic stimulation of quiescent fibroblasts. Similarly, early G1 phase ERK activity is dispensable for entry into S phase. Moreover, if cyclin D1 is expressed ectopically, ERK activity becomes dispensable throughout the G1 phase. In addition to its effect on cyclin D1, ERK activity is thought to contribute to the down-regulation of p27kip1. We found that this effect is restricted to late G1/S phase. Mechanistic analysis showed that the ERK effect on p27kip1 is mediated by Skp2 and is secondary to its effect on cyclin D1. Our results emphasize the importance of mid-G1 phase ERK activity and resolve primary versus secondary ERK targets within the G1 phase cyclin-dependent kinases.

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Interferon-α delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinases

Hepatology ◽

10.1053/jhep.2001.21749 ◽

2001 ◽

Vol 33 (2) ◽

pp. 346-356 ◽

Cited By ~ 56

Author(s):

D Murphy

Keyword(s):

Hepatocellular Carcinoma ◽

S Phase ◽

Carcinoma Cells ◽

Human Hepatocellular Carcinoma ◽

Interferon Α ◽

Hepatocellular Carcinoma Cells ◽

Cyclin Dependent Kinases ◽

Phase Progression ◽

Human Hepatocellular Carcinoma Cells

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Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase

Biochemical Society Transactions ◽

10.1042/bst0390600 ◽

2011 ◽

Vol 39 (2) ◽

pp. 600-605 ◽

Cited By ~ 20

Author(s):

Vasundhara M. Navadgi-Patil ◽

Peter M. Burgers

Keyword(s):

Cell Cycle ◽

Replication Stress ◽

Replication Initiation ◽

Initiation Factor ◽

Ataxia Telangiectasia Mutated ◽

Checkpoint Activation ◽

Dna Replication Initiation ◽

G2 Phase ◽

Two Phases ◽

Human Rad9

Mec1 [ATR (ataxia telangiectasia mutated- and Rad3-related) in humans] is the principle kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. The heterotrimeric checkpoint clamp, 9-1-1 (checkpoint clamp of Rad9, Rad1 and Hus1 in humans and Ddc1, Rad17 and Mec3 in S. cerevisiae; Ddc1-Mec3-Rad17) and the DNA replication initiation factor Dpb11 (human TopBP1) are the two known activators of Mec1. The 9-1-1 clamp functions in checkpoint activation in G1- and G2-phase, but its employment differs between these two phases of the cell cycle. The Ddc1 (human Rad9) subunit of the clamp directly activates Mec1 in G1-phase, an activity identified only in S. cerevisiae so far. However, in G2-phase, the 9-1-1 clamp activates the checkpoint by two mechanisms. One mechanism includes direct activation of Mec1 by the unstructured C-terminal tail of Ddc1. The second mech-anism involves the recruitment of Dpb11 by the phosphorylated C-terminal tail of Ddc1. The latter mechanism is highly conserved and also functions in response to replication stress in higher eukaryotes. In S. cerevisiae, however, both the 9-1-1 clamp and the Dpb11 are partially redundant for checkpoint activation in response to replication stress, suggesting the existence of additional activators of Mec1.

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Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1

International Journal of Molecular Sciences ◽

10.3390/ijms22168796 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8796

Author(s):

Antonio Galarreta ◽

Pablo Valledor ◽

Oscar Fernandez-Capetillo ◽

Emilio Lecona

Keyword(s):

Dna Replication ◽

Genomic Stability ◽

S Phase ◽

Replication Initiation ◽

Cancer Therapies ◽

Replication Machinery ◽

Post Translational Modification ◽

Aaa Atpase ◽

Dna Replication Initiation ◽

Set Up

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.

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ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin

The Journal of Cell Biology ◽

10.1083/jcb.201206084 ◽

2012 ◽

Vol 200 (1) ◽

pp. 31-44 ◽

Cited By ~ 64

Author(s):

Kyoo-young Lee ◽

Haiqing Fu ◽

Mirit I. Aladjem ◽

Kyungjae Myung

Keyword(s):

Dna Replication ◽

Molecular Mechanisms ◽

S Phase ◽

Nuclear Antigen ◽

Spatial Regulation ◽

Replication Factory ◽

G2 Phase ◽

Phase Progression ◽

Temporal And Spatial ◽

Proliferating Cell

Temporal and spatial regulation of the replication factory is important for efficient DNA replication. However, the underlying molecular mechanisms are not well understood. Here, we report that ATAD5 regulates the lifespan of replication factories. Reduced expression of ATAD5 extended the lifespan of replication factories by retaining proliferating cell nuclear antigen (PCNA) and other replisome proteins on the chromatin during and even after DNA synthesis. This led to an increase of inactive replication factories with an accumulation of replisome proteins. Consequently, the overall replication rate was decreased, which resulted in the delay of S-phase progression. Prevalent detection of PCNA foci in G2 phase cells after ATAD5 depletion suggests that defects in the disassembly of replication factories persist after S phase is complete. ATAD5-mediated regulation of the replication factory and PCNA required an intact ATAD5 ATPase domain. Taken together, our data imply that ATAD5 regulates the cycle of DNA replication factories, probably through its PCNA-unloading activity.

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Cyclin-Dependent Kinases and S Phase Control in Mammalian Cells

Cell Cycle ◽

10.4161/cc.2.4.468 ◽

2003 ◽

Vol 2 (4) ◽

pp. 315-323 ◽

Cited By ~ 107

Author(s):

Richard A. Woo ◽

Randy Y.C. Poon

Keyword(s):

Mammalian Cells ◽

Phase Control ◽

S Phase ◽

Cyclin Dependent Kinases

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The Intra-S-Phase Checkpoint Affects both DNA Replication Initiation and Elongation: Single-Cell and -DNA Fiber Analyses

Molecular and Cellular Biology ◽

10.1128/mcb.02278-06 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5806-5818 ◽

Cited By ~ 148

Author(s):

Jennifer A. Seiler ◽

Chiara Conti ◽

Ali Syed ◽

Mirit I. Aladjem ◽

Yves Pommier

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Human Colon ◽

S Phase ◽

Replication Initiation ◽

Dna Double Strand Breaks ◽

Dna Replication Initiation ◽

Nucleotide Incorporation ◽

Replication Foci ◽

S Phase Checkpoint

ABSTRACT To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.

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The limiting DNA replication initiation factors Sld2 and Sld3 influence origin efficiency independent of origin firing time

10.1101/384644 ◽

2018 ◽

Author(s):

Kelsey L. Lynch ◽

Elizabeth X. Kwan ◽

Gina M. Alvino ◽

Bonita J. Brewer ◽

M.K. Raghuraman

Keyword(s):

Rdna Locus ◽

S Phase ◽

Replication Initiation ◽

Yeast Genome ◽

Phase Duration ◽

Origin Firing ◽

Dna Replication Initiation ◽

Initiation Factors ◽

Origin Activation ◽

Time Of Origin

AbstractChromosome replication in Saccharomyces cerevisiae is initiated from roughly 300 origins that are regulated both by DNA sequence and by the limited abundance of four trans-acting initiation proteins (Sld2, Sld3, Dpb11 and Dbf4, collectively called “SSDD”). We set out to determine how the association of Sld2 or Sld3 at origins contributes to time of origin activation and/or origin efficiency using auxin-induced protein degradation to further decrease their abundance. Depleting cells of either factor slows growth rate, increases S-phase duration, and causes viability defects, without activating the S phase checkpoint. Chr XII is uniquely unstable with breakage occurring specifically within the rDNA locus. The efficiency of the rDNA origin is decreased while the onset of replication initiation is unchanged. We found that origin efficiency is reduced uniformly across the unique portions of the yeast genome. We conclude that the abundance of Sld2 and Sld3 contribute primarily to origin efficiency.

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