scholarly journals Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

2006 ◽  
Vol 26 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Guochun Jiang ◽  
Aziz Sancar
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Excision Repair ◽  
Dna Lesions ◽  
Human Cells ◽  
Dna Damage Checkpoint ◽  
Damage Site ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
Transcription Complexes

ABSTRACT We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

scholarly journals Phosphorylation of the Budding Yeast 9-1-1 Complex Is Required for Dpb11 Function in the Full Activation of the UV-Induced DNA Damage Checkpoint

2008 ◽  
Vol 28 (15) ◽  
pp. 4782-4793 ◽  
Author(s):  
Fabio Puddu ◽  
Magda Granata ◽  
Lisa Di Nola ◽  
Alessia Balestrini ◽  
Gabriele Piergiovanni ◽  
...  
Keyword(s):  
Dna Damage ◽  
Budding Yeast ◽  
Dna Damage Checkpoint ◽  
Checkpoint Activation ◽  
Checkpoint Response ◽  
Checkpoint Proteins ◽  
M Phase ◽  
Mutant Cells ◽  
Full Activation

ABSTRACT Following genotoxic insults, eukaryotic cells trigger a signal transduction cascade known as the DNA damage checkpoint response, which involves the loading onto DNA of an apical kinase and several downstream factors. Chromatin modifications play an important role in recruiting checkpoint proteins. In budding yeast, methylated H3-K79 is bound by the checkpoint factor Rad9. Loss of Dot1 prevents H3-K79 methylation, leading to a checkpoint defect in the G1 phase of the cell cycle and to a reduction of checkpoint activation in mitosis, suggesting that another pathway contributes to Rad9 recruitment in M phase. We found that the replication factor Dpb11 is the keystone of this second pathway. dot1Δ dpb11-1 mutant cells are sensitive to UV or Zeocin treatment and cannot activate Rad53 if irradiated in M phase. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with the phosphorylated 9-1-1 complex, leading to Mec1-dependent phosphorylation of Rad9. Dpb11 is also phosphorylated after DNA damage, and this modification is lost in a nonphosphorylatable ddc1-T602A mutant. Finally, we show that, in vivo, Dpb11 cooperates with Dot1 in promoting Rad9 phosphorylation but also contributes to the full activation of Mec1 kinase.

scholarly journals Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

2007 ◽  
Vol 27 (19) ◽  
pp. 6852-6862 ◽  
Author(s):  
Aimin Peng ◽  
Andrea L. Lewellyn ◽  
James L. Maller
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Nuclear Dna ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Checkpoint Signaling ◽  
Checkpoint Response ◽  
Egg Extracts ◽  
Cycle Checkpoint ◽  
Damaged Dna

ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

scholarly journals MEC3, MEC1, and DDC2Are Essential Components of a Telomere Checkpoint Pathway Required for Cell Cycle Arrest during Senescence in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (8) ◽  
pp. 2626-2638 ◽  
Author(s):  
Shinichiro Enomoto ◽  
Lynn Glowczewski ◽  
Judith Berman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Telomerase Activity ◽  
Cellular Level ◽  
Dna Damage Checkpoint ◽  
Average Growth ◽  
Progressive Decline ◽  
Checkpoint Response ◽  
Essential Components

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, andDDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1,RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.

Exploring DNA damage responses in human cells with recombinant adenoviral vectors

2007 ◽  
Vol 26 (11) ◽  
pp. 899-906
Author(s):  
Melissa G. Armelini ◽  
Keronninn M. Lima-Bessa ◽  
Maria Carolina N. Marchetto ◽  
Alysson R. Muotri ◽  
Vanessa Chiganças ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Adenoviral Vectors ◽  
Dna Lesions ◽  
Human Cells ◽  
Polymerase Eta ◽  
Cell Culture Systems

Recombinant adenoviral vectors provide efficient means for gene transduction in mammalian cells in vitro and in vivo. We are currently using these vectors to transduce DNA repair genes into repair deficient cells, derived from xeroderma pigmentosum (XP) patients. XP is an autosomal syndrome characterized by a high frequency of skin tumors, especially in areas exposed to sunlight, and, occasionally, developmental and neurological abnormalities. XP cells are deficient in nucleotide excision repair (affecting one of the seven known XP genes, xpa to xpg) or in DNA replication of DNA lesions (affecting DNA polymerase eta, xpv). The adenovirus approach allows the investigation of different consequences of DNA lesions in cell genomes. Adenoviral vectors carrying several xp and photolyases genes have been constructed and successfully tested in cell culture systems and in vivo directly in the skin of knockout model mice. This review summarizes these recent data and proposes the use of recombinant adenoviruses as tools to investigate the mechanisms that provide protection against DNA damage in human cells, as well as to better understand the higher predisposition of XP patients to cancer. Human & Experimental Toxicology (2007) 26, 899—906

scholarly journals Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

2008 ◽  
Vol 28 (24) ◽  
pp. 7402-7413 ◽  
Author(s):  
Sergey Alekseev ◽  
Martijn S. Luijsterburg ◽  
Alex Pines ◽  
Bart Geverts ◽  
Pierre-Olivier Mari ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dynamic Binding ◽  
Damage Site ◽  
Damage Recognition ◽  
Mutagenic Effects ◽  
Damaged Dna

ABSTRACT Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.

scholarly journals Yeast Securin is trafficked through the endomembrane system to regulate cell cycle arrest during the DNA damage response

2019 ◽  
Author(s):  
Brenda R. Lemos ◽  
David P. Waterman ◽  
James E. Haber
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Ubiquitin Proteasome System ◽  
Dna Damage Checkpoint ◽  
Transport Pathway ◽  
Checkpoint Response ◽  
Cycle Arrest ◽  
Chromatid Segregation ◽  
Ubiquitin Proteasome

AbstractThe yeast securin protein, Pds1, belongs to a class of highly conserved eukaryotic proteins that regulate the timing of chromatid segregation during mitosis by inhibiting separase, Esp1. During the metaphase to anaphase transition Pds1 is degraded by the ubiquitin-proteasome system in the nucleus, unleashing Esp1’s protease activity to cleave cohesin surrounding sister chromatids. In response to DNA damage Pds1 is phosphorylated and stabilized, stalling mitotic progression to preserve genomic integrity. In addition, during the DNA damage checkpoint response, securin and separase are partially localized in the vacuole. Here we genetically dissect the requirements for securin’s vacuolar localization and find that it is dependent on the Alkaline Phosphatase (ALP) endosomal transport pathway but not autophagy. Blocking retrograde traffic between the Golgi and the endoplasmic reticulum, by inhibiting COPI vesicle transport, drives Pds1 into the vacuole, whereas inhibiting antegrade transport by disrupting COPII-mediated traffic, results in the unexpected loss of Pds1 and the extinction of DNA damage-induced cell cycle arrest. We report that the induction of ER stress, either genetically or by treating cells with dithiothreitol, is sufficient to extinguish the DNA damage checkpoint, suggesting crosstalk between these two pathways. These data highlight new ways in which Pds1 and Esp1 are regulated during a DNA damage induced G2/M arrest and its requirement in the maintenance of the DNA damage checkpoint response.

scholarly journals DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites

2017 ◽  
Vol 217 (2) ◽  
pp. 527-540 ◽  
Author(s):  
Shalaka Chitale ◽  
Holger Richly
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Histone H4 ◽  
Damage Site ◽  
Nucleotide Excision ◽  
Lesion Recognition

Ultraviolet (UV) irradiation triggers the recruitment of DNA repair factors to the lesion sites and the deposition of histone marks as part of the DNA damage response. The major DNA repair pathway removing DNA lesions caused by exposure to UV light is nucleotide excision repair (NER). We have previously demonstrated that the endoribonuclease DICER facilitates chromatin decondensation during lesion recognition in the global-genomic branch of NER. Here, we report that DICER mediates the recruitment of the methyltransferase MMSET to the DNA damage site. We show that MMSET is required for efficient NER and that it catalyzes the dimethylation of histone H4 at lysine 20 (H4K20me2). H4K20me2 at DNA damage sites facilitates the recruitment of the NER factor XPA. Our work thus provides evidence for an H4K20me2-dependent mechanism of XPA recruitment during lesion recognition in the global-genomic branch of NER.

scholarly journals XPA: DNA Repair Protein of Significant Clinical Importance

2020 ◽  
Vol 21 (6) ◽  
pp. 2182 ◽  
Author(s):  
Lucia Borszéková Pulzová ◽  
Thomas A. Ward ◽  
Miroslav Chovanec
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Excision Repair ◽  
Protein A ◽  
Clinical Importance ◽  
Chemotherapeutic Agents ◽  
Dna Lesions ◽  
Response To Treatment ◽  
Damage Site

The nucleotide excision repair (NER) pathway is activated in response to a broad spectrum of DNA lesions, including bulky lesions induced by platinum-based chemotherapeutic agents. Expression levels of NER factors and resistance to chemotherapy has been examined with some suggestion that NER plays a role in tumour resistance; however, there is a great degree of variability in these studies. Nevertheless, recent clinical studies have suggested Xeroderma Pigmentosum group A (XPA) protein, a key regulator of the NER pathway that is essential for the repair of DNA damage induced by platinum-based chemotherapeutics, as a potential prognostic and predictive biomarker for response to treatment. XPA functions in damage verification step in NER, as well as a molecular scaffold to assemble other NER core factors around the DNA damage site, mediated by protein–protein interactions. In this review, we focus on the interacting partners and mechanisms of regulation of the XPA protein. We summarize clinical oncology data related to this DNA repair factor, particularly its relationship with treatment outcome, and examine the potential of XPA as a target for small molecule inhibitors.

scholarly journals Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Yi-Chih Tsai ◽  
Yi-Hsiang Wang ◽  
Yin-Chang Liu
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Mammalian Cells ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Subsequent Treatment ◽  
Dna Lesions ◽  
Gap Filling ◽  
Repair Synthesis ◽  
Damage Site

UVC irradiation-caused DNA lesions are repaired in mammalian cells solely by nucleotide excision repair (NER), which consists of sequential events including initial damage recognition, dual incision of damage site, gap-filling, and ligation. We have previously shown that gap-filling during the repair of UV-induced DNA lesions may be delayed by a subsequent treatment of oxidants or prooxidants such as hydrogen peroxide, flavonoids, and colcemid. We considered the delay as a result of competition for limiting protein/enzyme factor(s) during repair synthesis between NER and base excision repair (BER) induced by the oxidative chemicals. In this report, using colcemid as oxidative stress inducer, we showed that colcemid-caused delay of gap-filling during the repair of UV-induced DNA lesions was attenuated by overexpression of PCNA but not ligase-I. PCNA knockdown, as expected, delayed the gap-filling of NER but also impaired the repair of oxidative DNA damage. Fen-1 knockdown, however, did not affect the repair of oxidative DNA damage, suggesting repair of oxidative DNA damage is not of long patch BER. Furthermore, overexpression of XRCC1 delayed the gap-filling, and presumably increase of XRCC1 pulls PCNA away from gap-filling of NER for BER, consistent with our hypothesis that delay of gap-filling of NER attributes the competition between NER and BER.

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