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First published online 14 September 2004
doi: 10.1242/jcs.01355

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After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

Institute of Molecular Biotechnology Jena, Beutenbergstr. 11, 07745 Jena, Germany

View larger version (38K): [in a new window]   Fig. 1. DNA damage after UV-A exposure measured by the alkaline and neutral Comet assay. , DNA single- and double-strand damages as detected by the alkaline Comet assay; , solely DNA double-strand breaks as measured with the neutral Comet assay. (A) Time- and fluence-dependent induction of DNA damage relative to the control level. (B) The removal of damages during repair. (Inset) Semi-logarithmic plot of the first 5 hours of repair. The values can be fitted by an exponential function. Comet specimens from non-exposed control cells (C), directly after 40 minutes of UV-A (D), and 5 hours post UV exposure of 1280 kJ/m2 (E). Bars, 20 µm. The data are pooled from four experiments with two slides analysed per experiment (60 cells each). The median from each experiment was calculated and the mean of each median value, together with the standard deviation, is plotted.

View larger version (44K): [in a new window]   Fig. 2. (A) Fluence-dependent induction of micronuclei. The formation of micronuclei is plotted against the irradiation time in minutes and total radiation fluence. An increase, according to Eqn 2, of the percentage of induced micronuclei proportional to the irradiation energy is demonstrated. Inset shows a typical micronucleus detected after UV-A exposure. (B) Histone H2AX phosphorylation in HaCaT cells after UV-A exposure. Inset left shows histone H2AX phoshorylation in control cells: only a weak diffuse signal is visible. Inset right shows cells after exposure to UVA irradiation in which a clear signal of -H2AX becomes visible. For the analysis, 2x200 cells were scored in three independent preparations, and the plot shows the mean values together with standard deviation. (C) Focus formation of DNA-PKcs. The frequency of cells with DNA-PKcs is plotted against the irradiation time and UV-A fluence. The insets show the distribution of DNA-PKcs in control cells (left) and in cells exposed to UV-A (right). (D) The frequency of cells showing Rad51 foci is plotted against the irradiation time and the energy of irradiation, revealing saturation at approximately 40%. Inset left shows Rad51 foci in non-irradiated cells: only a few weak signals are detectable. By contrast, inset right shows that after UV-A exposure many cells show strong signals. (E) Response curves, normalised to the maximum response: dose dependencies of the data presented in A-D. The steepest increase is detected for -H2AX, whereas the lowest detected is for micronuclei formation.

View larger version (43K): [in a new window]   Fig. 3. Cell-cycle dependence of -H2AX, DNA-PKcs and Rad51 foci after irradiation with 960 kJ/m2. (A) Frequencies of -H2AX, DNA-PKcs and Rad51 foci in cell-cycle-synchronised cells. The Rad51 foci are mainly formed in G2 phase, whereas there are only a small number of cells with Rad51 foci in G1. By contrast, the DNA-PKcs and -H2AX foci are not related to the cell-cycle stage. (B-D) Sample micrographs of Rad51 foci, DNA-PKcs foci and H2AX foci in synchronised cells. The Rad51 foci are prevalent in S and G2 phases, whereas the -H2AX (right) and DNA-PKcs (middle) are not. Each experiment has been independently repeated and 2x200 cells were scored. (E) Flow-cytometric analysis of cell-cycle position after propidium iodide staining of total DNA.

View larger version (53K): [in a new window]   Fig. 4. Interaction of DSB repair proteins. (A,B) Close interaction is found e.g. for Rad51/Rad52 and Mre11/Rad50, where not only optical co-localisation can be found, but also the FRET effect is positive and shows close spatial proximity. (C) For Rad51/Rad52 and Mre11/Rad50, molecular interaction can be seen after UV-A exposure as demonstrated by the inducible complexes seen during Co-IP. (D-J) Optical co-localisation as merged images (arrows point to co-localisation spots) in G2-enriched cultures. These examples show that on some, but not all, DSB sites the DSB proteins from both pathways are present, but they do not have direct molecular interaction. The complete data from these experiments are summarised in Table 1.

View larger version (60K): [in a new window]   Fig. 5. Temporal sequence of focus and complex formation in unsynchronised cells. (A) The complex formation of Rad51 (black), Mre11 (red), -H2AX (green), DNA-PKcs (purple) and XRCC4 (blue) was studied after UV-A exposure. The -H2AX foci were formed first, quickly followed by DNA-PKcs. By contrast, the Rad51 and the XRCC4 foci are slower. The fast formation of -H2AX foci is followed by a fast reduction of the number of cells with foci. By contrast, the slowly formed complexes are stable over hours. Mre11 foci show an intermediate formation velocity and also an intermediate stability. (B) Also, the formation of protein-protein complexes has been studied in response to UVA. The damage signalling -H2AX foci were co-localised with the Rad51 foci, but the resulting complex was not formed immediately probably due to the slow building kinetic of the Rad51 foci. By contrast, the Rad50/Mre11 complex was formed immediately after exposure, whereas the Rad51/Rad52 complex is formed more slowly. (C) Sample images of focus formation and reduction after UV-A exposure. 1 hour post-irradiation the Rad51 foci are clearly visible; 5 hours post-irradiation the foci have mostly disassembled. (D) Sample micrograph of the optical co-localisation of Rad51 with -H2AX during and after UV-A exposure.

View larger version (29K): [in a new window]   Fig. 6. Spatial interaction pattern between proteins from the NHEJ and the HRR pathway. The black arrows indicate the closest interaction, direct protein-protein interaction detected by Co-IP. The grey arrows mark close spatial (but not direct protein-protein) interaction detected by the FRET effect. The dotted lines indicate optical co-localisation, which means proximity in the range of less than 200 nm (but on the same individual DSB site).

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