Cell cycle inertia underlies a bifurcation in cell fates after DNA damage

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.

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Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis

PLoS ONE ◽

10.1371/journal.pone.0129438 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0129438 ◽

Cited By ~ 20

Author(s):

Walter Georgescu ◽

Alma Osseiran ◽

Maria Rojec ◽

Yueyong Liu ◽

Maxime Bombrun ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Tracking ◽

Time Lapse ◽

Tracking Algorithms ◽

Damage Response

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NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.09.035 ◽

2011 ◽

Vol 414 (1) ◽

pp. 123-128 ◽

Cited By ~ 8

Author(s):

Burcu Erbaykent-Tepedelen ◽

Besra Özmen ◽

Lokman Varisli ◽

Ceren Gonen-Korkmaz ◽

Bilge Debelec-Butuner ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Cell Lines ◽

Cancer Cell ◽

Dna Damage Response ◽

Prostate Cancer Cell ◽

S Phase ◽

Prostate Cancer Cell Lines ◽

Damage Response ◽

Phase Entry

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Overall Cdk activity modulates the DNA damage response in mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200903033 ◽

2009 ◽

Vol 187 (6) ◽

pp. 773-780 ◽

Cited By ~ 42

Author(s):

Antonio Cerqueira ◽

David Santamaría ◽

Bárbara Martínez-Pastor ◽

Miriam Cuadrado ◽

Oscar Fernández-Capetillo ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Cyclin Dependent Kinase ◽

Genome Maintenance ◽

Cycle Progression ◽

Damage Response ◽

Dna Break ◽

Specific Contribution

In response to DNA damage, cells activate a phosphorylation-based signaling cascade known as the DNA damage response (DDR). One of the main outcomes of DDR activation is inhibition of cyclin-dependent kinase (Cdk) activity to restrain cell cycle progression until lesions are healed. Recent studies have revealed a reverse connection by which Cdk activity modulates processing of DNA break ends and DDR activation. However, the specific contribution of individual Cdks to this process remains poorly understood. To address this issue, we have examined the DDR in murine cells carrying a defined set of Cdks. Our results reveal that genome maintenance programs of postreplicative cells, including DDR, are regulated by the overall level of Cdk activity and not by specific Cdks.

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Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014048 ◽

2020 ◽

Vol 295 (50) ◽

pp. 17169-17186

Author(s):

Mysore S. Veena ◽

Santanu Raychaudhuri ◽

Saroj K. Basak ◽

Natarajan Venkatesan ◽

Parameet Kumar ◽

...

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Dna Damage ◽

Cell Growth ◽

Cell Lines ◽

Cancer Cell ◽

Dna Damage Response ◽

S Phase ◽

Cervical Cancer Cell ◽

Damage Response

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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Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽

10.1182/blood.v104.11.723.723 ◽

2004 ◽

Vol 104 (11) ◽

pp. 723-723

Author(s):

Alexandra Sobeck ◽

Stacie Stone ◽

Bendert deGraaf ◽

Vincenzo Costanzo ◽

Johan deWinter ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

S Phase ◽

Dependent Manner ◽

Core Complex ◽

Damage Response ◽

Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

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Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525620113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3676-E3685 ◽

Cited By ~ 8

Author(s):

Nicholas A. Willis ◽

Chunshui Zhou ◽

Andrew E. H. Elia ◽

Johanne M. Murray ◽

Antony M. Carr ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Dna Damage Response ◽

Cellular Response ◽

Genome Stability ◽

Biological Significance ◽

S Phase ◽

Damage Response

The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase–specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

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Genome-Wide Dynamic Evaluation of the UV-Induced DNA Damage Response

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401417 ◽

2020 ◽

Vol 10 (9) ◽

pp. 2981-2988

Author(s):

Erica Silva ◽

Manuel Michaca ◽

Brenton Munson ◽

Gordon J Bean ◽

Philipp A Jaeger ◽

...

Keyword(s):

Dna Damage ◽

Ultraviolet Radiation ◽

Dna Damage Response ◽

Protective Factor ◽

Protein Complexes ◽

Time Lapse ◽

Time Dependent ◽

Radiation Response ◽

Transient Effects ◽

Damage Response

Abstract Genetic screens in Saccharomyces cerevisiae have allowed for the identification of many genes as sensors or effectors of DNA damage, typically by comparing the fitness of genetic mutants in the presence or absence of DNA-damaging treatments. However, these static screens overlook the dynamic nature of DNA damage response pathways, missing time-dependent or transient effects. Here, we examine gene dependencies in the dynamic response to ultraviolet radiation-induced DNA damage by integrating ultra-high-density arrays of 6144 diploid gene deletion mutants with high-frequency time-lapse imaging. We identify 494 ultraviolet radiation response genes which, in addition to recovering molecular pathways and protein complexes previously annotated to DNA damage repair, include components of the CCR4-NOT complex, tRNA wobble modification, autophagy, and, most unexpectedly, 153 nuclear-encoded mitochondrial genes. Notably, mitochondria-deficient strains present time-dependent insensitivity to ultraviolet radiation, posing impaired mitochondrial function as a protective factor in the ultraviolet radiation response.

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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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DNA Repair and Longevity: A Possible Link in the Mouse Hematopoietic System.

Blood ◽

10.1182/blood.v104.11.1272.1272 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1272-1272

Author(s):

Jeff L. Yates ◽

Hartmut Geiger ◽

Gary Van Zant

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Dna Synthesis ◽

Dna Damage Response ◽

Bone Marrow Cells ◽

S Phase ◽

Chromosome 7 ◽

Brdu Incorporation ◽

Damage Response ◽

Marrow Cells

Abstract DNA repair efficiency has been postulated to play a role in aging-associated phenotypes as well as in the generation of a variety of cancers. This is especially pertinent in highly proliferative tissues such as the lymphohematopoietic system, since the stem and progenitor compartments are responsible for maintaining proliferative demands within a restricted range for the lifetime of an individual. Hydroxyurea (HU) is a chemotherapeutic drug that targets DNA synthesis by inhibiting the synthesis of the nucleotide substrate resulting in stalled replication forks and single- and double-stranded breaks (DSBs) in the DNA. Recently, our lab has mapped a locus on mouse chromosome 7 that is involved in both organismal lifespan determination and HU sensitivity of bone marrow stem and progenitor cells in the HU-sensitive (25.9% killing), short-lived (540 days) DBA/2J (D2) and HU-insensitive (11.8% killing), long-lived (816 days) C57Bl/6J (B6) strains of mouse. To confirm that this locus is responsible for hydroxyurea sensitivity we generated congenic mice where the locus-containing interval was moved from B6 to D2 (D2. B6 chr. 7) and vice versa (B6. D2 chr. 7). When these animals were treated with HU it was found that the D2 locus imparts a high killing phenotype (38.0%) and the B6 locus confers a low killing phenotype (−4.2%). Using a flow cytometry-based in vivo Bromodeoxyuridine (BrdU) incorporation assay, we measured the recovery of DNA synthesis in the bone marrow in D2 and B6 mice after IP injection of HU (2mg/g). We first determined that DNA synthesis was completely inhibited within 15 minutes of injection and persisted for at least 3 hours in both mouse strains. At 4 hours, bone marrow cells of both strains began to incorporate BrdU, with B6 recovery more rapid than D2, 2.9+/−.5 vs. 7.9+/−3.9 percent BrdU positive cells (p=.01), respectively. Because HU has been used in the past to synchronize cells in G0/G1 and to measure cells in S phase, it was expected that BrdU incorporation would re-initiate within the G0/G1 compartment of cells. Indeed, bone marrow cells from D2 mice incorporated BrdU exclusively within the G0/G1 population. Surprisingly it was found that cells from B6 mice that had an S phase content of DNA prior to HU survived the insult and began to synthesize DNA. It was concluded that B6 bone marrow might have a more robust DNA damage response than that of D2. To study the DNA damage response in the bone marrow we treated mice with HU followed by BrdU and stained the bone marrow cells with an anti-BrdU antibody and an antibody to gamma-H2AX (gH2AX), a histone variant that becomes phosphorylated in the vicinity of DNA DSBs. In both D2 and B6 bone marrow cells it was shown that maximal gH2AX phosphorylation occurred within 1 hour and only occurred in the BrdU+ fraction of the bone marrow cells. Thus it can be concluded that HU causes DNA damage and these two strains of mouse differ in their response due in part to a locus on chromosome 7. Current studies are aimed at identifying the gene(s) of interest in the congenic interval, which include Tfpt and Prkcc.

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