RESUMO
The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.
Assuntos
Cromatina/química , Dano ao DNA/genética , Reparo do DNA/genética , DNA de Neoplasias/química , DNA de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transativadores/química , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Difusão , Humanos , Modelos Genéticos , Modelos Estatísticos , Osteossarcoma/química , Osteossarcoma/genética , Ligação ProteicaRESUMO
A simple model of homogenous chromatin distribution in HeLa-cell nuclei suggests that the track of an energetic ion hits 30 nm chromatin fibers with a mean distance of 0.55 mum. To test this assumption, living HeLa-cells were irradiated at the irradiation setup of the ion microprobe SNAKE using the ion beams provided by the Munich 14 MV tandem accelerator. After irradiation, the distribution of 53BP1 protein foci was studied by immunofluorescence. The observed 53BP1 distribution along the tracks of 29 MeV (7)Li ions and 24 MeV (12)C ions differed significantly from the expectations resulting from the simple chromatin model, suggesting that the biological track structure is determined by cell nuclear architecture with higher order organisation of chromatin.
Assuntos
Cromatina/química , Cromatina/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/química , DNA/química , DNA/efeitos da radiação , Simulação por Computador , Proteínas de Ligação a DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Células HeLa , Íons Pesados , Humanos , Transferência Linear de Energia , Modelos Químicos , Modelos Moleculares , Doses de RadiaçãoRESUMO
The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV (16)O ions, the adapted set-up permits an irradiation accuracy of 0.91 microm (full width at half maximum) in the x-direction and 1.60 microm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.
