scholarly journals Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

1999 ◽  
Vol 10 (11) ◽  
pp. 3661-3674 ◽  
Author(s):  
Colin P. C. De Souza ◽  
Xiang S. Ye ◽  
Stephen A. Osmani
Keyword(s):  
Dna Damage ◽  
Tyrosine Phosphorylation ◽  
Complex Function ◽  
Ectopic Expression ◽  
S Phase ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
G2 Checkpoint ◽  
Low Concentrations ◽  
Dna Rereplication

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 inAspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions,uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea.uvsB is shown to encode a rad3/ATRhomologue, whereas uvsD displays homology torad26, which has only previously been identified inSchizosaccharomyces pombe. uvsB rad3 anduvsD rad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

scholarly journals Combined Inactivation of Pocket Proteins and APC/CCdh1 by Cdk4/6 Controls Recovery from DNA Damage in G1 Phase

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 550
Author(s):  
Indra A. Shaltiel ◽  
Alba Llopis ◽  
Melinda Aprelia ◽  
Rob Klompmaker ◽  
Apostolos Menegakis ◽  
...  
Keyword(s):  
Dna Damage ◽  
Normal Cell ◽  
S Phase ◽  
G1 Phase ◽  
Anaphase Promoting Complex ◽  
Inhibitory Effects ◽  
Pocket Proteins ◽  
Cyclin Dependent Kinases ◽  
Independent Functions ◽  
Phase Entry

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.

scholarly journals G2 Checkpoint Control: Checking in to the Last Resort for DNA Damage

2004 ◽  
Vol 3 (3) ◽  
pp. 314-316 ◽  
Author(s):  
Jeffrey R. Jackson ◽  
Bin Bing Zhou
Keyword(s):  
Dna Damage ◽  
Checkpoint Control ◽  
G2 Checkpoint

scholarly journals A Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

1999 ◽  
Vol 19 (8) ◽  
pp. 5535-5547 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Etsuko Matsui ◽  
Min Kwan Cho ◽  
Hiroyuki Kumagai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Fission Yeast ◽  
Kinase Activity ◽  
S Phase ◽  
Regulatory Subunit ◽  
Checkpoint Control ◽  
Regulatory Subunits ◽  
Hydroxyurea Treatment ◽  
S Phase Checkpoint

ABSTRACT Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+ is identical to rad35+ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

scholarly journals Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

1999 ◽  
Vol 10 (7) ◽  
pp. 2199-2208 ◽  
Author(s):  
Thomas E. Patterson ◽  
Elizabeth B. Albrecht ◽  
Paul Nurse ◽  
Shelley Sazer ◽  
George R. Stark
Keyword(s):  
Dna Damage ◽  
Gene Amplification ◽  
Genetic Background ◽  
S Phase ◽  
Checkpoint Control ◽  
Wild Type ◽  
Extrachromosomal Elements ◽  
Resistant Strains ◽  
Chromosome I ◽  
Selection Of

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing bothsod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications ofsod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

scholarly journals Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage

2009 ◽  
Vol 35 (3) ◽  
pp. 327-339 ◽  
Author(s):  
Neil Johnson ◽  
Dongpo Cai ◽  
Richard D. Kennedy ◽  
Shailja Pathania ◽  
Mansi Arora ◽  
...  
Keyword(s):  
Dna Damage ◽  
S Phase ◽  
Checkpoint Control ◽  
S Phase Checkpoint

scholarly journals Dosage Suppressors of pds1 Implicate Ubiquitin-Associated Domains in Checkpoint Control

2001 ◽  
Vol 21 (6) ◽  
pp. 1997-2007 ◽  
Author(s):  
Duncan J. Clarke ◽  
Guillaume Mondesert ◽  
Marisa Segal ◽  
Bonnie L. Bertolaet ◽  
Sanne Jensen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Spindle Assembly ◽  
S Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Checkpoint Control ◽  
Checkpoint Signaling ◽  
Ubiquitin System ◽  
Dependent Cell ◽  
S Phase Checkpoint

ABSTRACT In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact withPDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37°C. Genetic and functional interactions of two suppressors are described. RAD23 andDDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

scholarly journals NF-κB Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition

1999 ◽  
Vol 19 (4) ◽  
pp. 2690-2698 ◽  
Author(s):  
Michael Hinz ◽  
Daniel Krappmann ◽  
Alexandra Eichten ◽  
Andreas Heder ◽  
Claus Scheidereit ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Ectopic Expression ◽  
Tumor Development ◽  
Growth Control ◽  
S Phase ◽  
Checkpoint Control ◽  
Cycle Progression

ABSTRACT Nuclear factor kappa B (NF-κB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-κB activity and cell cycle regulation. NF-κB was found to stimulate transcription of cyclin D1, a key regulator of G1checkpoint control. Two NF-κB binding sites in the human cyclin D1 promoter conferred activation by NF-κB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1phase were controlled by NF-κB. Moreover, inhibition of NF-κB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-κB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-κB superrepressor (IκBαΔN) could be rescued by ectopic expression of cyclin D1. Thus, NF-κB contributes to cell cycle progression, and one of its targets might be cyclin D1.

scholarly journals Schizosaccharomyces pombe Cells Lacking the Amino-Terminal Catalytic Domains of DNA Polymerase Epsilon Are Viable but Require the DNA Damage Checkpoint Control

2001 ◽  
Vol 21 (14) ◽  
pp. 4495-4504 ◽  
Author(s):  
Wenyi Feng ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Checkpoint Control ◽  
Dna Polymerase Epsilon ◽  
Catalytic Domains ◽  
C Terminus ◽  
N Terminus ◽  
Polymerase Epsilon

ABSTRACT In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol ɛ) is encoded bycdc20 + and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol ɛ that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol ɛ (cdc20 Δ N-term ) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability ofcdc20 Δ N-term cells is dependent on expression ofrad3 +, hus1 +, andchk1 +, three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol ɛ, cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly forcdc20 Δ N-term cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol ɛ is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

scholarly journals Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 63
Author(s):  
Fanghua Li ◽  
Emil Mladenov ◽  
Rositsa Dueva ◽  
Martin Stuschke ◽  
Beate Timmermann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Current View ◽  
Checkpoint Control ◽  
Dna Double Strand Breaks ◽  
Independent Manner ◽  
G2 Checkpoint ◽  
G1 Checkpoint ◽  
G2 Phase ◽  
In Cells

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

scholarly journals DNA Damage Triggers p21WAF1-dependent Emi1 Down-Regulation That Maintains G2 Arrest

2009 ◽  
Vol 20 (7) ◽  
pp. 1891-1902 ◽  
Author(s):  
Jinho Lee ◽  
Jin Ah Kim ◽  
Valerie Barbier ◽  
Arun Fotedar ◽  
Rati Fotedar
Keyword(s):  
Dna Damage ◽  
Cell Cycle Progression ◽  
Control Cell ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
G2 Arrest ◽  
Down Regulation ◽  
Cycle Progression ◽  
M Phase

Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21WAF1, a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21WAF1 in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21+/+ cells stably arrest in G2, whereas p21−/− cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21+/+ cells. Inactivation of APC in irradiated p21+/+ cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21−/− cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage.

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