scholarly journals Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

1997 ◽  
Vol 17 (3) ◽  
pp. 1425-1433 ◽  
Author(s):  
S E Lee ◽  
R A Mitchell ◽  
A Cheng ◽  
E A Hendrickson
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Strand Break ◽  
S Phase ◽  
Dependent Manner ◽  
Severe Combined Immune Deficiency ◽  
Dsb Repair ◽  
G2 Checkpoint ◽  
G2 Phase

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.

scholarly journals Involvement of Brca1 in S-Phase and G2-Phase Checkpoints after Ionizing Irradiation

2001 ◽  
Vol 21 (10) ◽  
pp. 3445-3450 ◽  
Author(s):  
Bo Xu ◽  
Seong-tae Kim ◽  
Michael B. Kastan
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cellular Responses ◽  
S Phase ◽  
Cell Cycle Checkpoints ◽  
Genetic Damage ◽  
Ionizing Irradiation ◽  
Direct Role ◽  
G2 Phase ◽  
Definitive Role

ABSTRACT Cell cycle arrests in the G1, S, and G2phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G2/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G2 arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G2/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G2/M checkpoint.

scholarly journals Transcription-Associated Recombination Is Dependent on Replication in Mammalian Cells

2007 ◽  
Vol 28 (1) ◽  
pp. 154-164 ◽  
Author(s):  
Ponnari Gottipati ◽  
Tobias N. Cassel ◽  
Linda Savolainen ◽  
Thomas Helleday
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
S Phase ◽  
Replication Forks ◽  
Higher Eukaryotes ◽  
Stalled Replication Forks ◽  
Gene Conversions

ABSTRACT Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

scholarly journals An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants.

1995 ◽  
Vol 15 (5) ◽  
pp. 2527-2535 ◽  
Author(s):  
R J Miltenberger ◽  
K A Sukow ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Phase Boundary ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Binding Activity ◽  
S Phase ◽  
Dependent Manner ◽  
Carbamoyl Phosphate ◽  
Dna Binding Activity ◽  
E Box

To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G1/S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at +55 to +75 (+55/+75) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed down-stream of the transcription start site. The same oligonucleotide conferred less G1/S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad +55/+75 element in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G1/S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G1/S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.

scholarly journals A UV-responsive G2 checkpoint in rodent cells.

1995 ◽  
Vol 15 (7) ◽  
pp. 3722-3730 ◽  
Author(s):  
D K Orren ◽  
L N Petersen ◽  
V A Bohr
Keyword(s):  
Cell Cycle ◽  
Uv Irradiation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Chinese Hamster Ovary Cells ◽  
S Phase ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Synchronized Cells ◽  
G2 Phase

We have studied the effect of UV irradiation on the cell cycle progression of synchronized Chinese hamster ovary cells. Synchronization of cells in S or G2 phase was accomplished by the development of a novel protocol using mimosine, which blocks cell cycle progression at the G1/S boundary. After removal of mimosine, cells proceed synchronously through the S and G2 phases, allowing manipulation of cells at specific points in either phase. Synchronization of cells in G1 was achieved by release of cells after a period of serum starvation. Cells synchronized by these methods were UV irradiated at defined points in G1, S, and G2, and their subsequent progression through the cell cycle was monitored. UV irradiation of G1-synchronized cells caused a dose-dependent delay in entry into S phase. Irradiation of S-phase-synchronized cells inhibited progression through S phase and then resulted in accumulation of cells for a prolonged interval in G2. Apoptosis of a subpopulation of cells during this extended period was noted. UV irradiation of G2-synchronized cells caused a shorter G2 arrest. The arrest itself and its duration were dependent upon the timing (within G2 phase) of the irradiation and the UV dose, respectively. We have thus defined a previously undescribed (in mammalian cells) UV-responsive checkpoint in G2 phase. The implications of these findings with respect to DNA metabolism are discussed.

scholarly journals DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G0 cells

2021 ◽  
Author(s):  
Jessica K Tyler ◽  
Faith C Fowler ◽  
Chelsea Peart ◽  
Bo-Ruei Chen ◽  
Barry Sleckman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Proliferating Cells ◽  
Dsb Repair ◽  
Extensive Resection ◽  
Key Factor ◽  
Dna End Resection ◽  
End Resection

DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs are predominately repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in proliferating cells at the G1 or G2 phase of the cell cycle was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.

Nuclear accumulation of ZFP36L1 is cell cycle-dependent and determined by a C-terminal serine-rich cluster

2020 ◽  
Vol 168 (5) ◽  
pp. 477-489
Author(s):  
Yuki Matsuura ◽  
Aya Noguchi ◽  
Shunsuke Sakai ◽  
Naoto Yokota ◽  
Hiroyuki Kawahara
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Rna Binding ◽  
S Phase ◽  
Nuclear Accumulation ◽  
Protein Accumulation ◽  
Dependent Manner ◽  
Rich Cluster ◽  
G2 Phase ◽  
Cycle Dependent

Abstract ZFP36L1 is an RNA-binding protein responsible for mRNA decay in the cytoplasm. ZFP36L1 has also been suggested as a nuclear-cytoplasmic shuttling protein because it contains a potential nuclear localization signal and a nuclear export signal. However, it remains unclear how the nuclear localization of ZFP36L1 is controlled. In this study, we provide evidence that the nuclear accumulation of ZFP36L1 protein is modulated in a cell cycle-dependent manner. ZFP36L1 protein accumulation in fractionated nuclei was particularly prominent in cells arrested at G1-/S-phase boundary, while it was downregulated in S-phase cells, and eventually disappeared in G2-phase nuclei. Moreover, forced nuclear targeting of ZFP36L1 revealed marked downregulation of this protein in S- and G2-phase cells, suggesting that ZFP36L1 can be eliminated in the nucleus. The C-terminal serine-rich cluster of ZFP36L1 is critical for the regulation of its nuclear accumulation because truncation of this probable disordered region enhanced the nuclear localization of ZFP36L1, increased its stability and abolished its cell cycle-dependent fluctuations. These findings provide the first hints to the question of how ZFP36L1 nuclear accumulation is controlled during the course of the cell cycle.

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 Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts.

1993 ◽  
Vol 121 (1) ◽  
pp. 101-111 ◽  
Author(s):  
M Pagano ◽  
R Pepperkok ◽  
J Lukas ◽  
V Baldin ◽  
W Ansorge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
S Phase ◽  
Gene Products ◽  
Related Gene ◽  
Cultured Human Fibroblasts ◽  
G2 Phase ◽  
The Relationship

In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.

scholarly journals The Meiosis-Specific Crs1 Cyclin Is Required for Efficient S-Phase Progression and Stable Nuclear Architecture

2021 ◽  
Vol 22 (11) ◽  
pp. 5483
Author(s):  
Luisa F. Bustamante-Jaramillo ◽  
Celia Ramos ◽  
Cristina Martín-Castellanos
Keyword(s):  
Cell Cycle ◽  
Translational Control ◽  
Cell Cycle Progression ◽  
Nuclear Architecture ◽  
Strand Break ◽  
Spindle Pole ◽  
S Phase ◽  
Cycle Progression ◽  
Single Wave ◽  
Phase Progression

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

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