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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.004523

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Activation of multiple DNA repair pathways by sub-nuclear damage induction methods

Christoffel Dinant^1,2,*, Martijn de Jager^2,*,, Jeroen Essers^2,3, Wiggert A. van Cappellen⁴, Roland Kanaar^2,3, Adriaan B. Houtsmuller^1, and Wim Vermeulen^2,

¹ Department of Pathology, Josephine Nefkens Institute, ErasmusMC, Rotterdam, The Netherlands
² Department of Cell Biology and Genetics, ErasmusMC, Rotterdam, The Netherlands
³ Department of Radiation Oncology, ErasmusMC, Rotterdam, The Netherlands
⁴ Department of Reproduction and Development, ErasmusMC, Rotterdam, The Netherlands

View larger version (78K): [in this window] [in a new window]   Fig. 1. The NER response to local pulsed 800 nm laser irradiation. (A) XPC-GFP-expressing cells were irradiated through a filter with UV-C light (spots indicated by arrows) and subsequently treated with 800 nm laser pulses (lines indicated by arrowheads). Induction of CPDs is shown by staining with the CPD antibody (red, right panel) both on UV-C and pulsed 800 nm locally irradiated areas. In both areas XPC-GFP accumulated (green, left panel). (B) XPC-GFP-expressing cells were treated as in panel A and stained for the presence of 6-4PPs (red, right panel). Pulsed 800 nm irradiation is able to induce 6-4PP-formation as shown by the lines indicated by the arrowheads (right panel). The bar graph indicates fluorescence intensities of the nucleus (1), pulsed 800 nm induced local damage (2) and UV-C induced local damage (3). (C) GFP-XPA accumulates to a limited extent on pulsed 800 nm induced damaged areas (arrowhead) compared to UV-C irradiated areas (arrow). The bar graph indicates fluorescence intensities of the nucleus (1), pulsed 800 nm induced local damage (2) and UV-C induced local damage (3).

View larger version (54K): [in this window] [in a new window]   Fig. 2. The DSB repair response to local pulsed 800 nm laser irradiation. (A) XPC-GFP-expressing cells were treated with pulsed 800 nm irradiation and presence of DSBs is shown by immunohistochemical staining with a PKcs antibody (lines in right panel indicated by arrowheads). The bright spots outside the damaged area in the right panel are nucleolar structures of unknown origin and it is unknown if they exist in a living cell as well. (B) XPC-GFP-expressing cells were treated as in panel A and stained for the presence of phosphorylated histone H2AX (H2AX). Accumulation of H2AX at areas irradiated by the pulsed 800 nm laser confirms the presence of DSBs (right panel, arrowheads). No accumulation of H2AX is found on UV-C irradiated spots (arrows). Earlier it was shown that phosphorylation of H2AX takes place after UV-C irradiation (Marti et al., 2006; O'Driscoll et al., 2003) and we have found this as well in other experiments (data not shown). It is possible that in this case the specific immunohistochemical staining of H2AX at UV-C damage was not strong enough to be detected over background signals. (C) Rad54-GFP expressing cells were irradiated in an area of approximately 5 µm2 with pulsed 800 nm light and the redistribution of fluorescence was studied in time. The boxed area is two times enlarged in the left bottom of both panels. Rad54-GFP accumulates in small foci at the damaged area. (D) YFP-MDC1(BRCT) expressing cells were irradiated in a rectangular line through the nucleus and fluorescence redistribution was followed in time. YFP-MDC1(BRCT) accumulates in large foci at the damaged area (boxed area, left panel).

View larger version (53K): [in this window] [in a new window]   Fig. 3. NER response to local Hoechst 33342 treatment + 405 nm irradiation. (A) XPC-GFP-expressing cells were irradiated through a filter with UV-C light (spots indicated by arrows), sensitized with Hoechst 33342 and subsequently locally treated with 405 irradiation in the nucleus (lines indicated by arrowheads). Induction of CPDs is shown by the CPD antibody staining (right panel) both on UV-C and H+405 treated areas. XPC-GFP accumulated on both areas irradiated through a filter with UV-C light (arrows) and irradiated with 405 nm in combination with Hoechst 33342 (arrowheads). (B) Treatment as in panel A, here cells were stained with an antibody that recognizes 6-4PPs (right panel). Surprisingly, no 6-4PP-staining can be detected on laser-irradiated areas (lines indicated by arrowheads), while the UV-C treated areas show a clear induction (arrows). The bar graph indicates fluorescence intensities of the nucleus (1), 405 nm combined with Hoechst 33342 treatment induced local damage (2) and UV-C induced local damage (3). (C) GFP-XPA accumulates to a low level on local damage induced by 405 nm laser irradiation in combination with Hoechst 33342 treatment (arrowhead) compared with local UV-C irradiated areas (arrow). The bar graph indicates fluorescence intensities of the nucleus (1), 405 nm combined with Hoechst 33342 treatment induced local damage (2) and UV-C induced local damage (3).

View larger version (58K): [in this window] [in a new window]   Fig. 4. DSB repair response to Hoechst 33342 + 405 nm damage. (A) YFP-MDC1(BRCT) expressing cells were incubated with Hoechst 33342 and irradiated in an area of approximately 5 µm2 (white box) with 405 nm laser-light and the fluorescence redistribution was followed in time. YFP-MDC1(BRCT) accumulates in a non-focal/homogenous pattern at the damaged area (right panel). (B) Ku-GFP expressing cells were incubated with Hoechst 33342 and irradiated with 405 nm light in a big (top cell) or small (lower cell) area in the nucleus (white boxes). The accumulation of Ku-GFP was followed in time (right panel). (C) PKcs does not accumulate at Hoechst 33342 + 405 nm treated sites (line in the nucleus indicated by arrowhead). The bright spots in the PKcs channel are described in Fig. 2A and are also found in cells that were not damaged. (D) Rad54-GFP expressing cells were treated with Hoechst 33342 and 405 nm irradiation (white boxes) and fluorescence redistribution was followed in time (right panel). Rad54-GFP accumulates in very low numbers at locally damaged areas in a non-focal/homogenous pattern. The bar graph indicates the fluorescence intensity in the nucleus (1) and at the locally damaged area (2).

View larger version (93K): [in this window] [in a new window]   Fig. 5. UV-C laser irradiation. (A) GFP-XPA expressing cells were irradiated with 266 nm either without (arrow) or with attenuation (arrowhead). GFP-XPA accumulates on both areas (green, left panel) whereas TUNEL (red, middle panel) only stains positive on the spot that was created without attenuation. (B) GFP-XPA expressing cells were irradiated by attenuated UV-C laser light (arrow). Presence of CPDs was shown by immunohistochemical staining with -CPD (red, middle panel). (C) Cells that were irradiated in G1 or G2 phase (homogeneous PCNA pattern, red, middle panel) show no accumulation of Rad54-GFP (green, left panel) 2 hours after irradiation (arrow). In cells that were irradiated in S phase (PCNA pattern in foci, red, middle panel) Rad54-GFP (green, left panel) accumulates at locally irradiated areas within 1 hour after irradiation (arrow).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Recruitment of DNA repair factors to various types of DNA damage. (A) XPC-GFP accumulates most efficiently in areas damaged with 266 nm laser light. The presence of Hoechst 33342 causes slower diffusion of XPC thus retarding its recruitment to DNA damage. (B) GFP-XPA also accumulates most efficiently in areas damaged with 266 nm laser light. GFP-XPA responds to a very small extent to pulsed 800 nm irradiation and 405 nm irradiation combined with Hoechst 33342. (C) YFP-MDC1(BRCT) is recruited quicker and in higher numbers to damaged areas in cells irradiated with 405 nm combined with Hoechst 33342 than in pulsed 800 nm-irradiated cells. (D) Rad54-GFP has a delayed response to pulsed 800 nm irradiation but it accumulates to a larger extent to these damages than to 405 nm combined with Hoechst 33342 irradiation.

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