156 results match your criteria: "Institute of Medical Radiation[Affiliation]"

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ.

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The p97-Ufd1-Npl4 ATPase complex is associated with the response to DNA damage and replication stress, but how its inactivation leads to manifestation of chromosome instability is unclear. Here, we show that p97-Ufd1-Npl4 has an additional direct role in the G2/M checkpoint. Upon DNA damage, p97-Ufd1-Npl4 binds CDC25A downstream of ubiquitination by the SCF-βTrCP ligase and facilitates its proteasomal degradation.

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It is becoming increasingly clear that cells exposed to ionizing radiation (IR) and other genotoxic agents (targeted cells) can communicate their DNA damage response (DDR) status to cells that have not been directly irradiated (bystander cells). The term radiation-induced bystander effects (RIBE) describes facets of this phenomenon, but its molecular underpinnings are incompletely characterized. Consequences of DDR in bystander cells have been extensively studied and include transformation and mutation induction; micronuclei, chromosome aberration and sister chromatid exchange formation; as well as modulations in gene expression, proliferation and differentiation patterns.

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Effects of chromatin decondensation on alternative NHEJ.

DNA Repair (Amst)

November 2013

Institute of Medical Radiation Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany.

In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]).

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Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing.

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Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells.

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Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated.

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In cells exposed to low linear energy transfer (LET) ionizing-radiation (IR), double-strand-breaks (DSBs) form within clustered-damage-sites (CDSs) from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that all DSBs form promptly and are immediately detected by the cellular DNA-damage-response (DDR) apparatus. However, there is evidence that the pool of DSBs detected by physical methods, such as pulsed-field gel electrophoresis (PFGE), comprises not only promptly forming DSBs (prDSBs) but also DSBs developing during lysis at high temperatures from thermally-labile sugar-lesions (TLSLs).

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In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ)--a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters.

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In cells exposed to ionizing radiation, double-strand breaks (DSBs) form within clustered damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that DSBs form promptly and are immediately detected and processed by the cellular DNA damage response apparatus. However, DSBs also form by delayed chemical conversion of thermally-labile sugar lesions (TLSL) to breaks.

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The deleterious effects of DNA damage on DNA replication have been demonstrated in many model systems and the mechanisms of the resulting inhibition have been a research focus for at least 40 years. Moreover, recent studies have identified several major components of the S-phase checkpoint, providing a mechanistic background for understanding the basis of inhibition of the initiation and elongation steps of DNA replication after DNA damage. Yet several aspects of the underlying biochemical mechanisms remain unresolved including the characterization of the enzymatic activities involved in checkpoint activation and the coordination of this process with DNA repair.

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In vitro rejoining of double strand breaks in genomic DNA.

Methods Mol Biol

January 2013

Institute of Medical Radiation Biology, Medical School, University of Duisburg-Essen, Essen, Germany.

Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end joining (NHEJ) pathways in higher eukaryotes, and have facilitated the functional characterization of several of its components including DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos, and Artemis. Nevertheless, there is evidence that as of yet uncharacterized repair factors may contribute to the efficiency of NHEJ, for example by modulating the activity of known factors. Also, the discovery of alternative pathways of NHEJ that function as backup to the classical DNA-PK-dependent pathway of NHEJ has added yet another dimension in the set of activities involved.

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Background: Mammalian cells employ at least two subpathways of non-homologous end-joining for the repair of ionizing radiation induced DNA double strand breaks: The canonical DNA-PK-dependent form of non-homologous end-joining (D-NHEJ) and an alternative, slowly operating, error-prone backup pathway (B-NHEJ). In contrast to D-NHEJ, which operates with similar efficiency throughout the cell cycle, B-NHEJ operates more efficiently in G2-phase. Notably, B-NHEJ also shows strong and as of yet unexplained dependency on growth activity and is markedly compromised in serum-deprived cells, or in cells that enter the plateau-phase of growth.

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Purpose: This investigation addresses the question of whether serum constituents have a modulating effect on the induction of bystander effects. It was carried out as part of an inter-laboratory comparison-exercise within the European Union-integrated project NOTE (Non-targeted effects of ionizing radiation). Serotonin is a neurotransmitter, which is present in the circulatory system and therefore in blood serum.

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Purpose: It has long been known that the proliferation status of a cell is a determinant of radiation response, and the available evidence implicates repair of DNA double-strand breaks (DSBs) in the underlying mechanism. Recent results have shown that a novel, highly error-prone pathway of nonhomologous end joining (NHEJ) operating as backup (B-NHEJ) processes DSBs in irradiated cells when the canonical, DNA-PK (DNA-dependent protein kinase)-dependent pathway of NHEJ (D-NHEJ) is compromised. Notably, B-NHEJ shows marked reduction in efficiency when D-NHEJ-deficient cells cease to grow and enter a plateau phase.

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Purpose: Radiation-induced adaptive response (AR) is a phenomenon of increased radioresistance mediated by a low priming dose of ionizing radiation (IR) applied prior to a higher challenging dose. We have previously shown that in mouse-embryo fibroblasts (MEF) and human A549 cells, AR is associated with enhanced repair of DNA double-strand breaks (DSB) by the DNA-PK-dependent pathway of non-homologous end-joining (D-NHEJ). Importantly, AR was 'transmitted' to non-irradiated bystander cells through transfer of medium from cells that had received a priming dose of IR.

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To study human cancer development, cell culture models for malignant transformation can be used. In 1999 Hahn and Coworkers introduced such a model system and established herewith a basis for research on human tumorigenesis. Primary human fibroblasts are sequentially transduced with defined genetic elements (hTERT, SV40 ER, and H-RasV12), resulting in four defined cell lines, whereby the last has a fully transformed phenotype.

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In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar-phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions.

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A defining characteristic of damage induced in the DNA by ionizing radiation (IR) is its clustered character that leads to the formation of complex lesions challenging the cellular repair mechanisms. The most widely investigated such complex lesion is the DNA double strand break (DSB). DSBs undermine chromatin stability and challenge the repair machinery because an intact template strand is lacking to assist restoration of integrity and sequence in the DNA molecule.

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Background: Impaired DNA repair mechanism is one of the main causes of tumor genesis. Study of intrinsic radiosensitivity of cancer patients in a non-target tissue (e.g.

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Purpose: The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK-dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4(-/-) mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation.

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Adaptive response (AR) is a term describing resistance to ionizing radiation-induced killing or formation of aberrant chromosomes that is mediated by pre-exposure to low ionizing radiation doses. The mechanism of AR remains elusive. Because cell killing and chromosome aberration formation derive from erroneous processing of DNA double-strand breaks (DSB), AR may reflect a modulation of DSB processing by nonhomologous end joining (NHEJ) or homologous recombination repair.

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In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity.

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Abstract Double-strand breaks (DSBs) can be generated in the DNA of mammalian cells when heat-labile sites induced by ionizing radiation within a clustered DNA damage site are thermally transformed to single-strand breaks (SSBs) and combine with other SSBs or heat-labile sites in the opposite DNA strand. When this thermal transformation of heat-labile sites to SSBs occurs during DNA preparation using high-temperature lysis, the DSB yield is overestimated by including DSBs not present in the tested cell. Low-temperature lysis avoids this artifact but shows slower repair kinetics for prompt DSBs than high-temperature lysis.

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Backup pathways of NHEJ in cells of higher eukaryotes: cell cycle dependence.

Radiother Oncol

September 2009

Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.

DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) in cells of higher eukaryotes are predominantly repaired by a pathway of non-homologous end joining (NHEJ) utilizing Ku, DNA-PKcs, DNA ligase IV, XRCC4 and XLF/Cernunnos (D-NHEJ) as central components. Work carried out in our laboratory and elsewhere shows that when this pathway is chemically or genetically compromised, cells do not shunt DSBs to homologous recombination repair (HRR) but instead use another form of NHEJ operating as a backup (B-NHEJ). Here I review our efforts to characterize this repair pathway and discuss its dependence on the cell cycle as well as on the growth conditions.

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