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PubMedDate not supplied

The emerging role of E2F-1 in the DNA damage response and checkpoint control.

Genotoxic stress triggers a myriad of cellular responses including cell cycle arrest, stimulation of DNA repair and apoptosis. A central role for the E2F-1 transcription factor in the DNA damage response pathway is gaining support. E2F-1 is phosphorylated by DNA damage responsive protein kinases, which leads to E2F-1 accumulation and the induction of apoptosis. In addition, emerging information suggests that E2F-1 may play a role in the detection and subsequent repair of damaged DNA.

Animals↗

PubMed2002

Hus1 acts upstream of chk1 in a mammalian DNA damage response pathway.

The evolutionarily conserved Hus1 proteins function in DNA damage response pathways that serve to maintain genomic stability. Cells lacking mouse Hus1 are hypersensitive to certain genotoxins, and we have explored the molecular basis for this defect by examining how Hus1 inactivation affects genotoxin-induced signaling events. p53 accumulation and activation in response to DNA damage appeared normal in Hus1 null cells. Likewise, Hus1 was dispensable for genotoxin-induced Chk2 phosphorylation. In contrast, Chk1 phosphorylation after genotoxic stress was greatly reduced in the absence of Hus1, but was restored in Hus1 null fibroblasts complemented by infection with a Hus1-expressing retrovirus. These results demonstrate that mouse Hus1 is required for a specific subset of DNA damage signaling events and functions to promote genotoxin-induced Chk1 phosphorylation.

Animals↗

PubMed2026

Vimentin loss inhibits DNA damage responses and promotes cancer cell survival.

Vimentin intermediate filaments are a hallmark of aggressive tumours and are widely linked to invasion and EMT, yet how vimentin-dependent mechanics shape genome maintenance and therapy response is unclear. Here we show that vimentin, particularly under compressive load, promotes DNA repair competence. In contrast, vimentin-negative cells show impaired DNA damage sensing and downstream signaling, ultimately leading to decreased apoptosis and promoting cell survival under genotoxic stress at the expense of genomic stability. Using controlled cell compression together with genetic and pharmacological perturbations, we find that loss of vimentin in glioblastoma cells limits the expression and activity of core repair pathways because of induced nuclear mechanical compression. Relieving nuclear compression restores DNA damage accumulation and repair kinetics. Functionally, suppression of DNA damage responses enhances survival after clinically relevant DNA-damaging treatments, including temozolomide, X-Ray radiation and cell invasion through tight spaces. These findings invert the prevailing view that vimentin's contribution to tumour progression stems from enhanced migration and identify a mechanochemical vimentin-nucleus axis that tunes DNA damage responses to favor therapy tolerance and genome evolution.

Journal Article↗

PubMed2006

The role of DNA damage response proteins at telomeres--an "integrative" model.

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.

Animals↗

PubMed2003

Induction of DNA damage responses by adozelesin is S phase-specific and dependent on active replication forks.

Adozelesin is an alkylating minor groove DNA binder that is capable of rapidly inhibiting DNA replication in treated cells through a trans-acting mechanism and preferentially arrests cells in S phase. It has been shown previously that in cells treated with adozelesin, replication protein A (RPA) activity is deficient, and the middle subunit of RPA is hyperphosphorylated. The adozelesin-induced RPA hyperphosphorylation can be blocked by the replicative DNA polymerase inhibitor, aphidicolin, suggesting that adozelesin-triggered cellular DNA damage responses require active DNA replication forks. These data imply that cellular DNA damage responses to adozelesin treatment are preferentially induced in S phase. Here, we show that RPA hyperphosphorylation, RPA intranuclear focalization, and gamma-H2AX intranuclear focalization induced by adozelesin treatment are all dependent on DNA replication fork progression, and focalization is only induced in S phase cells. These findings are similar to those seen with the S phase-specific DNA-damaging agent, camptothecin. Conversely, all three DNA damage responses are independent of either S phase or replication fork progression when induced by treatment with the DNA strand scission agent, C-1027. Furthermore, we demonstrate that adozelesin-induced RPA and gamma-H2AX intranuclear foci appear to colocalize within the nuclei of S phase cells.

Aminoglycosides↗

PubMed2005

DNA damage response and Ku80 function in the vertebrate embryo.

Cellular responses to DNA damage reflect the dynamic integration of cell cycle control, cell-cell interactions and tissue-specific patterns of gene regulation that occurs in vivo but is not recapitulated in cell culture models. Here we describe use of the zebrafish embryo as a model system to identify determinants of the in vivo response to ionizing radiation-induced DNA damage. To demonstrate the utility of the model we cloned and characterized the embryonic function of the XRCC5 gene, which encodes Ku80, an essential component of the nonhomologous end joining pathway of DNA repair. After the onset of zygotic transcription, Ku80 mRNA accumulates in a tissue-specific pattern, which includes proliferative zones of the retina and central nervous system. In the absence of genotoxic stress, zebrafish embryos with reduced Ku80 function develop normally. However, low dose irradiation of these embryos during gastrulation leads to marked apoptosis throughout the developing central nervous system. Apoptosis is p53 dependent, indicating that it is a downstream consequence of unrepaired DNA damage. Results suggest that nonhomologous end joining components mediate DNA repair to promote survival of irradiated cells during embryogenesis.

Amino Acid Sequence↗

PubMed2005

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

Apoptosis↗

PubMed2003

MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways.

Forkhead-homology-associated (FHA) domains function as protein-protein modules that recognize phosphorylated serine/threonine motifs. Interactions between FHA domains and phosphorylated proteins are thought to have essential roles in the transduction of DNA damage signals; however, it is unclear how FHA-domain-containing proteins participate in mammalian DNA damage responses. Here we report that a FHA-domain-containing protein-mediator of DNA damage checkpoint protein 1 (MDC1; previously known as KIAA0170)--is involved in DNA damage responses. MDC1 localizes to sites of DNA breaks and associates with CHK2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of CHK2. Furthermore, MDC1 is phosphorylated in an ATM/CHK2-dependent manner after DNA damage, suggesting that MDC1 may function in the ATM-CHK2 pathway. Consistent with this hypothesis, suppression of MDC1 expression results in defective S-phase checkpoint and reduced apoptosis in response to DNA damage, which can be restored by the expression of wild-type MDC1 but not MDC1 with a deleted FHA domain. Suppression of MDC1 expression results in decreased p53 stabilization in response to DNA damage. These results suggest that MDC1 is recruited through its FHA domain to the activated CHK2, and has a critical role in CHK2-mediated DNA damage responses.

Adaptor Proteins, Signal Transducing↗

PubMed2003

ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response.

The ATM protein kinase regulates the DNA damage response by phosphorylating proteins involved in cell cycle checkpoints and DNA repair. We report here on the function of the predicted leucine zipper (LZ) motif, and sequences adjacent to this, in regulating ATM activity. The predicted LZ sequence was deleted from ATM, generating ATMDeltaLZ, and expressed in an ATM-negative AT cell line. ATM increased cell survival following exposure to ionizing radiation, whereas expression of ATMDeltaLZ failed to increase cell survival. ATMDeltaLZ retained in vitro kinase activity, but was unable to phosphorylate p53 in vivo. Leucine zippers mediate homo- and heterodimerization of proteins. However, the predicted LZ of ATM did not mediate the formation of ATM dimers. We examined if the predicted LZ of ATM was a dominant-negative inhibitor of ATM function in SW480 cells. Expression of amino acids 769-1436 of ATM, including the predicted LZ, sensitized SW480 cells to ionizing radiation, but did not inhibit ATM's kinase activity or its ability to phosphorylate Brca1. Further, this dominant-negative activity was not dependent on the predicted LZ domain. The central region of the ATM protein therefore contains multiple sequences which regulate cell survival following DNA damage.

Amino Acid Motifs↗

PubMed2007

p53 modulation of the DNA damage response.

The tumor suppressor p53 plays a central role in the DNA damage response. After exposure to genotoxic stress, p53 can both positively and negatively regulate cell fate. Initially, p53 promotes cell survival by inducing cell cycle arrest, DNA repair, and other pro-survival pathways. However, when cells accumulate DNA damage or demonstrate aberrant growth, p53 can direct the elimination of damaged cells. In this review, we will discuss the transcriptional-dependent and -independent roles of p53 in regulating the DNA damage response.

Animals↗

PubMed2005

Telomere biology: integrating chromosomal end protection with DNA damage response.

Telomeres play the key protective role at chromosomes. Many studies indicate that loss of telomere function causes activation of DNA damage response. Here, we review evidence supporting interdependence between telomere maintenance and DNA damage response and present a model in which these two pathways are combined into a single mechanism for protecting chromosomal integrity. Proteins directly involved in telomere maintenance and DNA damage response include Ku, DNA-PKcs, RAD51D, PARP-2, WRN and RAD50/MRE11/NBS1 complex. Since most of these proteins participate in the repair of DNA double-strand breaks (DSBs), this was perceived by many authors as a paradox, given that telomeres function to conceal natural DNA ends from mechanisms that detect and repair DSBs. However, we argue here that the key function of one particular DSB protein, Ku, is to prevent or control access of telomerase, the enzyme that synthesises telomeric sequences, to both internal DSBs and natural chromosomal ends. This view is supported by observations that Ku has a high affinity for DNA ends; it acts as a negative regulator of telomerase and that telomerase itself can target internal DSBs. Ku then directs other DSB repair/telomere maintenance proteins to either repair DSBs at internal chromosomal sites or prevent uncontrolled elongation of telomeres by telomerase. This model eliminates the above paradox and provides a testable scenario in which the role of DSB repair proteins is to protect chromosomal integrity by balancing repair activities and telomere maintenance. In our model, a close association between telomeres and different DNA damage response factors is not an unexpected event, but rather a logical result of chromosomal integrity maintenance activities.

Animals↗

PubMed2007

The contribution of the DNA damage response to neuronal viability.

Neurons are extremely active cells and metabolize up to 20% of the oxygen that was consumed by the organism. Despite their highly oxygenic metabolism, neuronal cells have a lower capacity to neutralize the reactive oxygen species (ROS) that they generate or to which they are exposed. High levels of ROS can lead to accumulation of damage to various cellular macromolecules. One of the cellular macromolecules highly affected by intracellular as well as extracellular insults is DNA. Neurons are also highly differentiated, postmitotic cells that cannot be replenished after disease or trauma. Since neurons are irreplaceable and should survive as long as the organism does, they need elaborate defense mechanisms to ensure their longevity. This review article mainly focuses on certain mechanisms that contribute to neuronal longevity, and concentrates on the DNA damage response in neuronal cells. The various mechanisms of DNA repair are briefly described, and focus is on those mechanisms that are activated in neuronal cells following DNA damage. Evidence is presented to show that proper DNA damage response is critically important, not just for normal neuronal development but throughout the entire life of any organism. Defective DNA damage response in older human age can generate neurodegenerative disorders such as Alzheimer's or Parkinson diseases.

Alzheimer Disease↗

PubMed2004

Coordination of DNA damage responses via the Smc5/Smc6 complex.

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.

Amino Acid Sequence↗

PubMed2005

NFBD1/Mdc1 mediates ATR-dependent DNA damage response.

Budding yeast Rad9 (scRad9) plays a central role in mediating Mec1-dependent phosphorylation by recruiting its downstream substrates. The human scRad9 orthologues 53BP1 and NFBD1 associate with ionizing radiation-induced foci (IRIF) at sites of DNA repair. RNAi-based gene silencing of 53BP1 or NFBD1 has shown impaired phosphorylation of SQ/TQ [ataxia-telangiectasia mutated/ATM and Rad3-related (ATM/ATR) substrates] at IRIF, intra-S, and G(2)-M checkpoints and has thereby revealed essential roles for 53BP1 and NFBD1 in the DNA damage signaling pathway. Whether 53BP1 and NFBD1 are required for activation of kinases and/or for recruitment of substrates at IRIF, however, is not clear. Here we show that both 53BP1 and NFBD1 are required for recruitment of ATR to DNA damage sites, as well as for ATR-dependent phosphorylation in response to DNA damage. NFBD1 is not required for ssDNA generation at DNA damage sites and is not recruited by replication protein A (RPA)-coated ssDNA. We therefore show that recruitment of NFBD1 and/or 53BP1, the factors downstream of H2AX, is independent of ssDNA generation and RPA coating, whereas both ssDNA and RPA coating play key roles in regulation of the ATR-dependent pathway. These novel findings help clarify where NFBD1 functions in DNA damage early responses.

Adaptor Proteins, Signal Transducing↗

PubMed2002

DNA damage responses protect xeroderma pigmentosum variant from UVC-induced clastogenesis.

Lack of DNA polymerase eta and the attendant defect in bypass replication of pyrimidine dimers induced in DNA by ultraviolet light (UV) underlie the enhanced mutagenesis and carcinogenesis observed in xeroderma pigmentosum variant (XP-V). We investigated whether diploid XP-V fibroblasts growing in culture are also more susceptible to UV-induced clastogenesis than normal human fibroblasts (NHF). This study utilized diploid fibroblasts immortalized by the ectopic expression of human telomerase. The cell lines displayed checkpoint responses to DNA damage comparable with those measured in the parental strains. Shortly after exposure to low doses of UVC (< or =4 J/m2), XP-V cells accumulated daughter strand gaps in excess of normal controls (>25-fold). Daughter strand gaps generated in UV-irradiated S phase cells are potential precursors of chromatid-type chromosomal aberrations. Nonetheless, chromatid-type chromosomal aberrations were only 1.5 to 2 times more abundant in XP-V than in NHF exposed to the same UVC dose. XP-V cells, however, displayed S phase delays at lower doses of UVC and for longer periods of time than NHF. These results support the hypothesis that aberrant DNA structures activate S phase checkpoint responses that increase the time available for postreplication repair. Alternatively, cells that cannot be properly repaired remain permanently arrested and never reach mitosis. These responses protect human cells from chromosomal aberrations, especially when other pathways, such as accurate lesion bypass, are lost.

Carcinogenicity Tests↗

PubMed1999

Expression of the HPV E7 oncoprotein mimics but does not evoke a p53-dependent cellular DNA damage response pathway.

Acute expression of the human papillomavirus E7 oncoprotein in preimmortal human fibroblasts induces changes in the abundances of multiple cellular regulatory proteins. These alterations include a destabilization of the retinoblastoma tumor suppressor protein pRB, stabilization of the tumor suppressor protein p53, and increases in the level of the cyclin-dependent kinase inhibitor p21(cip1). Since the HPV E7 oncoproteins can interfere with several cell cycle checkpoints and similar alterations in the levels of pRB, p53, and p21(cip1) are also observed in a p53-dependent response to DNA damage, we investigated whether E7 expression triggers this signal transduction pathway. The results demonstrate that E7-mediated destabilization of pRB does not require p53 activity and is independent of the ability of E7 to induce apoptosis. Moreover, E7-mediated increases in p21(cip1) levels are largely p53-independent and involve stabilization of the p21(cip1) protein. In contrast the decreases in pRB expression in response to DNA damage involve transcriptional downregulation of RB gene expression.

Apoptosis↗

PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗

PubMed2004

Phosphatases join kinases in DNA-damage response pathways.

An inappropriate imbalance of kinase and phosphatase activities could be deleterious to cellular processes such as proliferation. Cellular responses to DNA damage use signal-transduction pathways involving phosphorylation events, and such modifications must be reversible to make these responses transient, rather than permanent, events. Three recent articles describe roles for two phosphatases in signaling pathways that are activated after DNA damage.

DNA Damage↗