ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

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A Role for NIPA in DNA Damage Response.

Blood ◽

10.1182/blood.v104.11.1265.1265 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1265-1265

Author(s):

Christine von Klitzing ◽

Florian Bassermann ◽

Stephan W. Morris ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Phosphorylation Site ◽

Genotoxic Stress ◽

S Phase ◽

Damage Response ◽

A Cell ◽

Cycle Dependent

Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

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Lack of CCAAT Enhancer Binding Protein Beta (C/EBPβ) in Uterine Epithelial Cells Impairs Estrogen-Induced DNA Replication, Induces DNA Damage Response Pathways, and Promotes Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.00872-09 ◽

2010 ◽

Vol 30 (7) ◽

pp. 1607-1619 ◽

Cited By ~ 16

Author(s):

Cyril Ramathal ◽

Indrani C. Bagchi ◽

Milan K. Bagchi

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Epithelial Cells ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

S Phase ◽

Uterine Epithelial Cells ◽

Cycle Arrest ◽

Damage Response

ABSTRACT Female mice lacking the transcription factor C/EBPβ are infertile and display markedly reduced estrogen (E)-induced proliferation of the uterine epithelial lining during the reproductive cycle. The present study showed that E-stimulated luminal epithelial cells of a C/EBPβ-null uterus are able to proceed through the G1 phase of the cell cycle before getting arrested in the S phase. This cell cycle arrest was accompanied by markedly reduced levels of expression of E2F3, an E2F family member, and a lack of nuclear localization of cyclin E, a critical regulator of cdk2. An increased nuclear accumulation of p27, an inhibitor of the cyclin E-cdk2 complex, was also observed for the mutant epithelium. Gene expression profiling of C/EBPβ-null uterine epithelial cells revealed that the blockade of E-induced DNA replication triggers the activation of several well-known components of the DNA damage response pathway, such as ATM, ATR, histone H2AX, checkpoint kinase 1, and tumor suppressor p53. The activation of p53 by ATM/ATR kinase led to increased levels of expression of p21, an inhibitor of G1-S-phase progression, which helps maintain cell cycle arrest. Additionally, p53-dependent mechanisms contributed to an increased apoptosis of replication-defective cells in the C/EBPβ-null epithelium. C/EBPβ, therefore, is an essential mediator of E-induced growth and survival of uterine epithelial cells of cycling mice.

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BK Polyomavirus Activates the DNA Damage Response To Prolong S Phase

Journal of Virology ◽

10.1128/jvi.00130-19 ◽

2019 ◽

Vol 93 (14) ◽

Cited By ~ 5

Author(s):

Joshua L. Justice ◽

Jason M. Needham ◽

Sunnie R. Thompson

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

S Phase ◽

Genome Replication ◽

Viral Production ◽

Viral Dna ◽

Bk Polyomavirus ◽

Damage Response ◽

Infected Cells

ABSTRACT BK polyomavirus (PyV) is a major source of kidney failure in transplant recipients. The standard treatment for patients with lytic BKPyV infection is to reduce immunosuppressive therapy, which increases the risk of graft rejection. PyVs are DNA viruses that rely upon host replication proteins for viral genome replication. A hallmark of PyV infection is activation of the DNA damage response (DDR) to prevent severe host and viral DNA damage that impairs viral production by an unknown mechanism. Therefore, we sought to better understand why BKPyV activates the DDR through the ATR and ATM pathways and how this prevents DNA damage and leads to increased viral production. When ATR was inhibited in BKPyV-infected primary kidney cells, severe DNA damage occurred due to premature Cdk1 activation, which resulted in mitosis of cells that were actively replicating host DNA in S phase. Conversely, ATM was required for efficient entry into S phase and to prevent normal mitotic entry after G2 phase. The synergistic activation of these DDR kinases promoted and maintained BKPyV-mediated S phase to enhance viral production. In contrast to BKPyV infection, DDR inhibition did not disrupt cell cycle control in uninfected cells. This suggests that DDR inhibitors may be used to specifically target BKPyV-infected cells. IMPORTANCE BK polyomavirus (BKPyV) is an emerging pathogen that reactivates in immunosuppressed organ transplant patients. We wanted to understand why BKPyV-induced activation of the DNA damage response (DDR) enhances viral titers and prevents host DNA damage. Here, we show that the virus activates the DNA damage response in order to keep the infected cells in S phase to replicate the viral DNA. The source of DNA damage was due to actively replicating cells with uncondensed chromosomes entering directly into mitosis when the DDR was inhibited in BKPyV-infected cells. This study clarifies the previously enigmatic role of the DDR during BKPyV infection by demonstrating that the virus activates the DDR to maintain the cells in S phase in order to promote viral replication and that disruption of this cell cycle arrest can lead to catastrophic DNA damage for the host.

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Tof1p Regulates DNA Damage Responses During S Phase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.2.567 ◽

2001 ◽

Vol 157 (2) ◽

pp. 567-577 ◽

Cited By ~ 2

Author(s):

Eric J Foss

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Double Mutant ◽

S Phase ◽

Damage Response ◽

Dna Damage Responses ◽

Dna Damage Response Pathway

Abstract A tof1 mutant was recovered in a screen aimed at identifying genes involved specifically in the S phase branch of the MEC1-dependent DNA damage response pathway. The screen was based on the observation that mutants missing this branch are particularly dependent on the cell cycle-wide branch and, therefore, on RAD9, for surviving DNA damage. tof1 and rad9 conferred synergistic sensitivity to MMS, UV, and HU, and the double mutant was incapable of slowing S phase in response to MMS, inducing RNR3 transcription in response to UV, and phosphorylating Rad53p in response to HU. TOF1's contribution to DNA damage response appeared to be restricted to S phase, since TOF1 did not contribute to UV-induced transcription during G1 or to the cdc13-1-induced block to anaphase in G2/M. I suggest a model in which Tof1p functions to link Mec1p with Rad53p.

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Elevated transcription of the p53 gene in early S-phase leads to a rapid DNA-damage response during S-phase of the cell cycle

APOPTOSIS ◽

10.1007/s10495-011-0623-z ◽

2011 ◽

Vol 16 (9) ◽

pp. 950-958 ◽

Cited By ~ 18

Author(s):

Paula Takahashi ◽

Amanda Polson ◽

David Reisman

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

P53 Gene ◽

S Phase ◽

Damage Response

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Schizosaccharomyces pombe Hst4 Functions in DNA Damage Response by Regulating Histone H3 K56 Acetylation

Eukaryotic Cell ◽

10.1128/ec.00379-07 ◽

2008 ◽

Vol 7 (5) ◽

pp. 800-813 ◽

Cited By ~ 30

Author(s):

Devyani Haldar ◽

Rohinton T. Kamakaka

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Schizosaccharomyces Pombe ◽

Histone Acetylation ◽

Dna Damage Response ◽

Damage Tolerance ◽

Histone H3 ◽

S Phase ◽

Dna Damage Tolerance ◽

Damage Response

ABSTRACT The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Δ mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Δ mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.

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Characterization of mec1Kinase-Deficient Mutants and of New Hypomorphic mec1Alleles Impairing Subsets of the DNA Damage Response Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.21.12.3913-3925.2001 ◽

2001 ◽

Vol 21 (12) ◽

pp. 3913-3925 ◽

Cited By ~ 70

Author(s):

Vera Paciotti ◽

Michela Clerici ◽

Maddalena Scotti ◽

Giovanna Lucchini ◽

Maria Pia Longhese

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Damage Response ◽

Cell Cycle Progression ◽

Uv Light ◽

Protein A ◽

S Phase ◽

Specific Cell ◽

Dna Damage Checkpoints ◽

Damage Response

ABSTRACT DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show thatmec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Δ cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G1 or during S phase. Conversely, an excess of Mec1kd inMEC1 cells does not abrogate the G2/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G1/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G2. The finding that these mutants, although indistinguishable frommec1Δ cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.

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