RESUMO
Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30â Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Ribossômico/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Ribossômico/genética , Humanos , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Transcrição Gênica/genéticaRESUMO
Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.
Assuntos
Carbono/química , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/química , Transferência Linear de Energia , Prótons , Rad51 Recombinase/química , Proteínas de Ciclo Celular/química , Reparo do DNA , Células HeLa , Humanos , Íons/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.
Assuntos
Cromossomos/efeitos da radiação , Dano ao DNA/efeitos da radiação , Inativação Gênica/efeitos da radiação , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Cromatina/química , Cromatina/genética , Cromossomos/química , Cromossomos/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Feminino , Imunofluorescência , Raios gama/efeitos adversos , Histonas/genética , Humanos , Metilação/efeitos da radiação , Microscopia Confocal , Osteossarcoma/genética , Osteossarcoma/patologia , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
During secondary immunoglobulin (Ig) diversification in vertebrates, the sequence of the variable region of Ig genes may be altered by templated or non-templated mechanisms. In both cases, cytidine deamination by activation-induced cytidine deaminase (AID) in the transcribed Ig loci leads to DNA lesions, which are repaired by conservative homologous recombination (HR) during Ig gene conversion, or by non-templated mutagenesis during somatic hypermutation. The molecular basis for the differential use of these two pathways in different species is unclear. While experimental ablation of HR in avian cells performing Ig gene conversion may promote a switch to somatic hypermutation, the activity of HR processes in intrinsically hypermutating mammalian cells has not been measured to date. Employing a functional HR assay in human germinal centre like B cell lines, we detect elevated HR activity that can be enhanced by transcription and AID. Products of such recombination events mostly arise through non-conservative HR pathways, while the activity of conservative HR is low to absent. Our results identify non-conservative HR as a novel DNA transaction pathway promoted by AID and suggest that somatic hypermutation in germinal centre B cells may be based on a physiological suppression of conservative HR.
Assuntos
Linfócitos B/imunologia , Citidina Desaminase/metabolismo , Recombinação Genética , Transcrição Gênica , Linhagem Celular , Linhagem Celular Tumoral , Centro Germinativo/imunologia , Humanos , Linfoma de Células B/genética , Hipermutação Somática de ImunoglobulinaRESUMO
The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Histonas/metabolismo , Rad51 Recombinase/metabolismo , Radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Imunofluorescência , Células HeLa , Humanos , NanoestruturasRESUMO
Radio (chemo) therapy is a crucial treatment modality for head and neck squamous cell carcinoma (HNSCC), but relapse is frequent, and the underlying mechanisms remain largely elusive. Therefore, novel biomarkers are urgently needed. Previously, we identified gains on 16q23-24 to be associated with amplification of the Fanconi anemia A (FancA) gene and to correlate with reduced progression-free survival after radiotherapy. Here, we analyzed the effects of FancA on radiation sensitivity in vitro, characterized the underlying mechanisms, and evaluated their clinical relevance. Silencing of FancA expression in HNSCC cell lines with genomic gains on 16q23-24 resulted in significantly impaired clonogenic survival upon irradiation. Conversely, overexpression of FancA in immortalized keratinocytes conferred increased survival accompanied by improved DNA repair, reduced accumulation of chromosomal translocations, but no hyperactivation of the FA/BRCA-pathway. Downregulation of interferon signaling as identified by microarray analyses, enforced irradiation-induced senescence, and elevated production of the senescence-associated secretory phenotype (SASP) appeared to be candidate mechanisms contributing to FancA-mediated radioresistance. Data of the TCGA HNSCC cohort confirmed the association of gains on 16q24.3 with FancA overexpression and impaired overall survival. Importantly, transcriptomic alterations similar to those observed upon FancA overexpression in vitro strengthened the clinical relevance. Overall, FancA amplification and overexpression appear to be crucial for radiotherapeutic failure in HNSCC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/radioterapia , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Amplificação de Genes , Neoplasias de Cabeça e Pescoço/radioterapia , Tolerância a Radiação/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Intervalo Livre de Doença , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genótipo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Estimativa de Kaplan-Meier , Queratinócitos/patologia , Queratinócitos/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Fatores de Tempo , Transfecção , Falha de Tratamento , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.
Assuntos
Poli(ADP-Ribose) Polimerases/metabolismo , Anticorpos de Domínio Único/imunologia , Ressonância de Plasmônio de Superfície/métodos , Especificidade de Anticorpos , Linhagem Celular , Sobrevivência Celular , DNA/genética , DNA/metabolismo , Epitopos/imunologia , Humanos , Imunoprecipitação , Imagem Molecular , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/imunologia , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Histone demethylases have recently gained interest as potential targets in cancer treatment and several histone demethylases have been implicated in the DNA damage response. We investigated the effects of siRNA-mediated depletion of histone demethylase Jarid1A (KDM5A, RBP2), which demethylates transcription activating tri- and dimethylated lysine 4 at histone H3 (H3K4me3/me2), on growth characteristics and cellular response to radiation in several cancer cell lines. In unirradiated cells Jarid1A depletion lead to histone hyperacetylation while not affecting cell growth. In irradiated cells, depletion of Jarid1A significantly increased cellular radiosensitivity. Unexpectedly, the hyperacetylation phenotype did not lead to disturbed accumulation of DNA damage response and repair factors 53BP1, BRCA1, or Rad51 at damage sites, nor did it influence resolution of radiation-induced foci or rejoining of reporter constructs. We conclude that the radiation sensitivity observed following depletion of Jarid1A is not caused by a deficiency in repair of DNA double-strand breaks.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/metabolismo , Tolerância a Radiação , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Acetilação , Proliferação de Células/efeitos da radiação , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Técnicas de Silenciamento de Genes , Genes Reporter , Células HeLa , Humanos , Lisina/metabolismo , Células MCF-7 , Plasmídeos/metabolismo , Tolerância a Radiação/efeitos da radiação , Radiação IonizanteRESUMO
The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination.
Assuntos
Proteínas de Ligação a DNA/deficiência , DNA , Recombinação Genética , Animais , Células CHO , Cricetinae , DNA/genética , DNA/metabolismo , Fibroblastos , Rad51 RecombinaseRESUMO
Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein (eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure.
Assuntos
Proteínas de Ligação a DNA/deficiência , DNA , Proteínas de Fluorescência Verde/fisiologia , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/deficiência , Recombinação Genética , Proteínas Supressoras de Tumor/deficiência , Animais , Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Células CHO , Proteínas de Ciclo Celular , Cromossomos/genética , Cricetinae , DNA/genética , DNA/metabolismo , DNA Recombinante , Fibroblastos/metabolismoRESUMO
The aim of this work is to review the uses of laser microirradiation and ion microbeam techniques within the scope of radiobiological research. Laser microirradiation techniques can be used for many different purposes. In a specific condition, through the use of pulsed lasers, cell lysis can be produced for subsequent separation of different analytes. Microsurgery allows for the identification and isolation of tissue sections, single cells and subcellular components, using different types of lasers. The generation of different types of DNA damage, via this type of microirradiation, allows for the investigation of DNA dynamics. Ion microbeams are important tools in radiobiological research. There are only a limited number of facilities worldwide where radiobiological experiments can be performed. In the beginning, research was mostly focused on the bystander effect. Nowadays, with more sophisticated molecular and cellular biological techniques, ion microirradiation is used to unravel molecular processes in the field of radiobiology. These include DNA repair protein kinetics or chromatin modifications at the site of DNA damage. With the increasing relevance of charged particles in tumour therapy and new concepts on how to generate them, ion microbeam facilities are able to address unresolved questions concerning particle tumour therapy.
Assuntos
Pesquisa Biomédica/métodos , Lasers de Gás , Microcirurgia/métodos , Radiobiologia/métodos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Relação Dose-Resposta à Radiação , HumanosRESUMO
Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.
Assuntos
Células/metabolismo , Células/efeitos da radiação , Imagem Molecular/instrumentação , Aceleradores de Partículas/instrumentação , Radiobiologia/instrumentação , Humanos , ÍonsRESUMO
The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0) after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1). Mdc1 accumulates faster (T(0) = 17 ± 2 s, τ(1) = 98 ± 11 s) than 53BP1 (T(0) = 77 ± 7 s, τ(1) = 310 ± 60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T(0) = 73 ± 16 s, τ(1) = 1050 ± 270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1) of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.
Assuntos
Dano ao DNA , Reparo do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Cinética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Raios UltravioletaRESUMO
BACKGROUND: Laser acceleration of protons and heavy ions may in the future be used in radiation therapy. Laser-driven particle beams are pulsed and ultra high dose rates of >109 Gy s⻹ may be achieved. Here we compare the radiobiological effects of pulsed and continuous proton beams. METHODS: The ion microbeam SNAKE at the Munich tandem accelerator was used to directly compare a pulsed and a continuous 20 MeV proton beam, which delivered a dose of 3 Gy to a HeLa cell monolayer within < 1 ns or 100 ms, respectively. Investigated endpoints were G2 phase cell cycle arrest, apoptosis, and colony formation. RESULTS: At 10 h after pulsed irradiation, the fraction of G2 cells was significantly lower than after irradiation with the continuous beam, while all other endpoints including colony formation were not significantly different. We determined the relative biological effectiveness (RBE) for pulsed and continuous proton beams relative to x-irradiation as 0.91 ± 0.26 and 0.86 ± 0.33 (mean and SD), respectively. CONCLUSIONS: At the dose rates investigated here, which are expected to correspond to those in radiation therapy using laser-driven particles, the RBE of the pulsed and the (conventional) continuous irradiation mode do not differ significantly.
Assuntos
Neoplasias/radioterapia , Apoptose , Ciclo Celular , Sobrevivência Celular , Citometria de Fluxo , Células HeLa , Íons Pesados , Humanos , Íons , Lasers , Microscopia de Fluorescência/métodos , Neoplasias/patologia , Aceleradores de Partículas , Prótons , Radioterapia/métodos , Eficiência Biológica Relativa , Fatores de Tempo , Raios XRESUMO
High-linear energy transfer (LET) ion irradiation of cell nuclei induces complex and severe DNA lesions, and foci of repair proteins are formed densely along the ion trajectory. To efficiently discriminate the densely distributed/overlapping foci along the ion trajectory, a focus recognition algorithm called FociPicker3D based on a local fraction thresholding technique was developed. We analyzed high-resolution 3D immunofluorescence microscopic focus images and obtained the kinetics and spatial development of γ-H2AX, 53BP1 and phospho-NBS1 foci in BJ1-hTERT cells irradiated with 55 MeV carbon ions and compared the results with the dynamics of double-strand break (DSB) distributions simulated using the PARTRAC model. Clusters consisting of several foci were observed along the ion trajectory after irradiation. The spatial dynamics of the protein foci supports that the foci clusters are not formed by neighboring foci but instead originate from the DSB cluster damage induced by high-LET radiations.
Assuntos
Carbono , Biologia Computacional/métodos , Dano ao DNA , Transferência Linear de Energia , Proteínas/metabolismo , Algoritmos , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Modelos Biológicos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Reprodutibilidade dos Testes , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
We have built an ion-microbeam for studies of the nuclear topography and kinetics of double-strand break repair at the single cell level. Here, we show that a first and a second, delayed single ion exposure at different nuclear sites led to comparable accumulations of phospho-ATM, gamma-H2AX and Mdc1 at both earlier (e) and later (l) microirradiated sites. In contrast, accumulations of 53BP1 and the recombination protein Rad51 were strongly reduced at l-sites. This apparent competition effect is accompanied by a reduced amount of 53BP1 in undamaged areas of the irradiated nuclei. We suggest that a critically limited pool size combined with strong binding at irradiated sites leads to the exhaustion of unbound factors freely roaming the nuclear space. The undersupply of these factors at l-sites requires in addition a long-lasting binding at e-sites or a weaker binding at l-sites. The observed effects suggest that DNA damage response at individual nuclear sites depends on the time course of damage load. This may have implications for therapeutic radiation treatments.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Reparo do DNA/efeitos da radiação , DNA/efeitos da radiação , Microscopia de Fluorescência/métodos , DNA/ultraestrutura , Relação Dose-Resposta à Radiação , Humanos , Doses de RadiaçãoRESUMO
Several proteins are known to form foci at DNA sites damaged by ionizing radiation. We study DNA damage response by immunofluorescence microscopy after microirradiation of cells with energetic ions. By using microirradiation, it is possible to irradiate different regions on a single dish at different time-points and to differentiate between cells irradiated earlier and later. This allows to directly compare immunofluorescence intensities in both subsets of cells with little systematic error because both subsets are cultivated and stained under identical conditions. In addition, by using irradiation patterns such as crossing lines, it is possible to irradiate individual cells twice and to differentiate between immunofluorescence signals resulting from the cellular response to the earlier and to the later irradiation event. Here, we describe the quantitative evaluation of immunofluorescence intensities after sequential irradiation.