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

Activation of multiple DNA repair pathways by sub-nuclear damage induction methods Research Article 2731 Activation of multiple DNA repair pathways by sub- nuclear damage induction methods 1,2, 2, ,‡ 2,3 4 2,3 Christoffel Dinant *, Martijn de Jager * , Jeroen Essers , Wiggert A. van Cappellen , Roland Kanaar , 1,§ 2,§ Adriaan B. Houtsmuller and Wim Vermeulen 1 2 3 Department of Pathology, Josephine Nefkens Institute, Department of Cell Biology and Genetics, Department of Radiation Oncology and Department of Reproduction and Development, ErasmusMC, Rotterdam, The Netherlands *These authors contributed equally to this work Present address: Physics of Life Processes, Leiden Institute of Physics (LION), Leiden University, Leiden, The Netherlands Authors for correspondence (e-mails: [email protected]; [email protected]) Accepted 30 May 2007 Journal of Cell Science 120, 2731-2740 Published by The Company of Biologists 2007 doi:10.1242/jcs.004523 Summary Live cell studies of DNA repair mechanisms are greatly cellular response to presensitization by Hoechst 33342 and enhanced by new developments in real-time visualization subsequent 405 nm irradiation were aberrant from those of repair factors in living cells. Combined with recent to every other DNA damaging method described here or in advances in local sub-nuclear DNA damage induction the literature. Whereas, application of low-dose 266 nm procedures these methods have yielded detailed laser irradiation induced only UV-specific DNA photo- information on the dynamics of damage recognition and lesions allowing the study of the UV-C-induced DNA repair. Here we analyze and discuss the various types of damage response in a user-defined area in cultured cells. DNA damage induced in cells by three different local damage induction methods: pulsed 800 nm laser irradiation, Hoechst 33342 treatment combined with 405 Supplementary material available online at nm laser irradiation and UV-C (266 nm) laser irradiation. http://jcs.biologists.org/cgi/content/full/120/15/2731/DC1 A wide variety of damage was detected with the first two methods, including pyrimidine dimers and single- and Key words: Pyrimidine dimers, Local DNA damage induction, double-strand breaks. However, many aspects of the Double-strand breaks, Living cells, DNA repair kinetics Introduction al., 2001; Katsumi et al., 2001; Mone et al., 2001; Nelms et al., The mammalian genome is protected against the continuous 1998; Volker et al., 2001) to focusing laser beams inside living stress of both exogenous and endogenous DNA damaging cell nuclei (Essers et al., 2006; Lukas et al., 2005). agents by a number of DNA damage response mechanisms, The kinetics of nucleotide excision repair (NER) have been including different DNA repair pathways. Unresolved DNA determined previously by irradiation of cultured cells through lesions may introduce mutations, which can lead to cancer a polycarbonate filter with UV-C light, either prior to or after (Mitchell et al., 2003). In addition, unrepaired damages may mounting on the microscope stage, and subsequently result in disturbed transcription and replication, which measuring the accumulation of repair proteins (Hoogstraten et eventually causes cell death contributing to aging. The severe al., 2002; Mone et al., 2004; Politi et al., 2005; Zotter et al., clinical consequences associated with hereditary disorders that 2006). In addition, alternative methods have been developed harbor defects in DNA repair systems underscore the where DNA damage is introduced by focused laser beams, at importance of efficient DNA repair (Bootsma and user-defined regions within the nucleus (Cremer et al., 1980; Hoeijmakers, 1994; Hoeijmakers, 2001). Lan et al., 2004; Meldrum et al., 2003; Walter et al., 2003). Genetic and biochemical analysis of repair processes have This approach allows great flexibility not only with respect to culminated in detailed mechanistic insight into the distinct position, but also size and shape of the local damage induced DNA repair processes. To study the interaction of the different in individual cells. DNA repair processes with each other and with other cellular Tuned localized intense laser irradiation with 365 nm light processes such as transcription and replication, spatiotemporal causes different types of DNA lesions ranging from oxidized analysis of different DNA repair systems in intact living cells base damage, single-strand breaks (SSBs) and up to double- is required and has been used extensively with the aid of GFP- strand breaks (DSBs) (Lan et al., 2004). Another powerful tagged repair factors (Essers et al., 2002b; Hoogstraten et al., method uses pulsed near infrared laser (multiphoton) 2002; Houtsmuller et al., 1999; Rademakers et al., 2003). technology. In this case two or three lower energy photons are Recently, DNA repair research has been boosted substantially absorbed simultaneously resulting in twice or three times the by the development of several methods to locally inflict DNA energy deposition. Meldrum et al. (Meldrum et al., 2003) damage in cultured living cells, enabling the direct applied this procedure using a pulsed 750 nm laser (with an visualization of GFP-tagged repair factors accumulating at the effective wavelength of 250 nm) and showed that this method sub-nuclear region where the damage is caused. These methods is able to create UV-like DNA lesions in living cells as shown range from irradiating partially shielded cells (Kannouche et by in situ immunostaining using antibodies against cyclobutane Journal of Cell Science 2732 Journal of Cell Science 120 (15) Table 1. Induced damages and protein accumulations: DSB and SSB repair Treatment TUNEL H2AX PKcs MDC1 Rad54 Ku70 PARP-1 Pulsed 800nm laser + + + + + + + Hoechst + 405 nm laser + + – +* +* + + † ‡ § § † UV-C – – –– – – – *DSB repair proteins that do not accumulate in foci but in a homogenous pattern; UV-C irradiation without attenuation resulted in positive TUNEL staining ‡ § and Ku-GFP accumulation; at higher UV-C doses H2AX accumulation can be found; accumulation of DSB repair proteins on UV-C damage is dependent on ongoing replication. Table 2. Induced damages and protein accumulations: pyrimidine dimers (CPDs). Recently, it has been shown that nucleotide excision repair (NER) with a pulsed near infrared laser DSBs are created as well Treatment CPD 6-4PP XPC XPA (Mari et al., 2006), indicating the broad spectrum of DNA lesions induced with this procedure. Pulsed 800 nm laser + + + + Hoechst+405 nm laser + – + + More indirect methods rely on local relatively low energy UV-C + + + + UV-A irradiation. These methods require cells to be pretreated with halogenated thymidine analogs such as BrdU or IdU, which are incorporated into DNA, and induce SSBs and DSBs when the cells are exposed to UV-A (Lukas et al., 2003; Tashiro et al., 2000). A variant of this method employs DNA- Response of the NER machinery to pulsed 800 nm binding dyes such as Hoechst either in combination with irradiation (Rogakou et al., 1999; Walter et al., 2003), or without To investigate the types of DNA damage created by pulsed near thymidine analogs (Bradshaw et al., 2005). Although a number infrared (NIR) laser irradiation, cells were subjected to high of these in situ local damage-inducing systems have been intensity 800 nm laser pulses. To provide an internal control applied to study DNA damage response mechanisms the for the immunofluorescent detection of pyrimidine dimers, we spectrum of DNA lesion induced by these procedures has not irradiated XPC-GFP expressing cells with UV-C light through been analyzed in great detail. a filter before irradiation with a NIR laser. Pulsed 800 nm laser We have systematically analyzed and compared three irradiation resulted in the formation of CPDs (Fig. 1A), as different procedures to locally inflict DNA damage in cultured reported previously (Meldrum et al., 2003). In addition to cells. We show that pulsed 800 nm irradiation introduces a CPDs, also 6-4PPs were formed (Fig. 1B; arrowheads). XPC- broad variety of DNA lesions at which proteins involved in GFP (Politi et al., 2005) accumulated in areas irradiated with different pathways accumulate. The combination of Hoechst a UV lamp through a micro-porous filter as well as areas 33342 incorporation and 405 nm irradiation induced a cellular irradiated with a pulsed 800 nm laser (Fig. 1A,B). GFP-XPA response that differed strongly from the response to other (Rademakers et al., 2003) also accumulated with both methods, damaging methods. In addition, we have developed a but there was a much stronger response to UV lamp irradiation microscope setting using focused UV-C (266 nm) laser than to pulsed 800 nm irradiation (Fig. 1C). irradiation, which induces predominantly UV-C-specific photolesions such as cyclobutane pyrimidine dimers (CPD) Response of the DSB repair machinery to pulsed and 6-4 photoproducts (6-4PP). 800 nm irradiation To determine whether DSBs are induced by a pulsed 800 nm Results laser we stained locally irradiated nuclei of XPC-deficient Experimental setup fibroblasts (XP4PA) expressing XPC-GFP (Politi et al., 2005) We have investigated local DNA damage induction in cultured with an antibody against phosphorylated DNA-PKcs (PKcs). living cells with confocal microscopy using lasers of different DNA-PKcs is the catalytic subunit of the DNA-dependent wavelengths: 800 nm, 405 nm and 266 nm. The types of protein kinase (DNA-PK), which is autophosphorylated in damages created with these methods and the assembly of response to ionizing radiation (Chan et al., 2002). The presence different repair proteins after local irradiation were first of PKcs suggested the formation of DSBs by a pulsed 800 analyzed using immunocytological procedures directed against nm laser (Fig. 2A). In addition, H2AX (Fig. 2B) and Ku80- lesions (CPD, 6-4PP and TUNEL) or the consequences of GFP (Mari et al., 2006) were also found at these sites, lesions (accumulation of phosphorylated H2AX, indicative of the presence of DSBs. Under similar conditions phosphorylated DNA-PKcs, PARP-1) as well as protein-GFP local UV-C irradiation through pores in a filter failed to induce fusions. The results of these studies are summarized in Tables DSBs as indicated by the absence of PKcs positive signal 1 and 2. In addition, the kinetics of protein interaction with (Table 1) and H2AX staining (Fig. 2B). DNA damage complexes were analyzed in living cells Rad54 is implicated in multiple steps of DSB repair through expressing fluorescently tagged repair factors involved in both homologous recombination (HR). Previous research has shown early and late steps of the reaction of both nucleotide excision that in response to DSB induction by ionizing radiation HR repair (involving XPC and XPA proteins) and DSB repair proteins accumulate in nuclear foci (Essers et al., 2002b; Rouse (MDC1and Rad54 proteins). All cell lines expressing GFP- or and Jackson, 2002; van Veelen et al., 2005a; van Veelen et al., YFP-tagged proteins have previously been characterized and 2005b). Accordingly, Rad54-GFP accumulated in a focal pattern published (see Materials and Methods section and references at the damaged area (Fig. 2C, right panel), similar to what has therein). been described for multiple HR proteins after DNA damage Journal of Cell Science Laser-induced DNA damage 2733 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 Fig. 2. The DSB repair response to local pulsed 800 nm laser intensities of the nucleus (1), pulsed 800 nm induced local damage irradiation. (A) XPC-GFP-expressing cells were treated with pulsed (2) and UV-C induced local damage (3). (C) GFP-XPA accumulates 800 nm irradiation and presence of DSBs is shown by to a limited extent on pulsed 800 nm induced damaged areas immunohistochemical staining with a PKcs antibody (lines in right (arrowhead) compared to UV-C irradiated areas (arrow). The bar panel indicated by arrowheads). The bright spots outside the damaged graph indicates fluorescence intensities of the nucleus (1), pulsed area in the right panel are nucleolar structures of unknown origin and 800 nm induced local damage (2) and UV-C induced local damage it is unknown if they exist in a living cell as well. (B) XPC-GFP- (3). 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 induction (Bekker-Jensen et al., 2006). Whereas HR is thought presence of DSBs (right panel, arrowheads). No accumulation of to be predominantly active during the S and G2 phases of the H2AX is found on UV-C irradiated spots (arrows). Earlier it was shown that phosphorylation of H2AX takes place after UV-C cell-cycle, we found accumulation of Rad54-GFP in virtually all irradiation (Marti et al., 2006; O’Driscoll et al., 2003) and we have cells. Similarly, Rad51 was found to accumulate at locally found this as well in other experiments (data not shown). It is possible damaged areas regardless of cell-cycle phase (Kim et al., 2005). that in this case the specific immunohistochemical staining of H2AX These observations suggest that part of the HR machinery is at UV-C damage was not strong enough to be detected over loaded onto DSBs in G1. However, this recruitment might not background signals. (C) Rad54-GFP expressing cells were irradiated reflect ongoing repair. Interestingly, the BRCT domain of MDC1 2 in an area of approximately 5 m with pulsed 800 nm light and the tagged with YFP [YFP-MDC1(BRCT)] also accumulated on redistribution of fluorescence was studied in time. The boxed area is pulsed 800 nm laser-induced damage, but with faster kinetics two times enlarged in the left bottom of both panels. Rad54-GFP and in much bigger foci than Rad54 (Fig. 2D, right panel, versus accumulates in small foci at the damaged area. (D) YFP- Fig. 2C, right panel). These large foci are likely indicative of MDC1(BRCT) expressing cells were irradiated in a rectangular line through the nucleus and fluorescence redistribution was followed in interaction between MDC1 and H2AX (Bekker-Jensen et al., time. YFP-MDC1(BRCT) accumulates in large foci at the damaged 2006). area (boxed area, left panel). To determine a dose of pulsed 800 nm radiation with which one specific repair pathway was induced and not another, we lowered the laser intensity. At slightly lower doses than used indicates that under the conditions used we did not observe above, both GFP-XPA (NER) and Rad54-GFP (HR) remained preferential formation of one type of lesion over the other by undetectable in the irradiated areas (data not shown). This changing the applied dose. Journal of Cell Science 2734 Journal of Cell Science 120 (15) pyrimidine dimer antibody staining before addition of Hoechst. After Hoechst 33342 treatment, a rectangular area of the nucleus of these cells was exposed to 405 nm irradiation. This resulted in abundant CPD formation (Fig. 3A) identical to pulsed 800 nm-induced damage (Fig. 1C). Remarkably, no 6- 4PPs were found (Fig. 3B). Apparently this method specifically induced minor helix distorting lesions such as CPDs but not the more severely helix distorting 6-4PPs. Similar to its response to pulsed 800 nm irradiation, XPC-GFP responded very strongly to these damages (Fig. 3A,B) but GFP-XPA accumulation was much less intense on Hoechst + 405 nm-irradiated areas than on UV lamp-irradiated areas (Fig. 3C). This suggests that XPC responds to a wider variety of lesions than only those typically repaired by NER. Response of the DSB repair machinery to Hoechst 33342 + 405 nm damage induction In Hoechst 33342-sensitized CHO9 cells locally irradiated at 405 nm, YFP-MDC1(BRCT) as well as the non-homologous end-joining (NHEJ)-specific Ku80-GFP quickly accumulated in the irradiated areas in very high numbers (Fig. 4A,B). See also Fig. S1 in supplementary material for colocalization of XPC-mCherry and YFP-MDC1(BRCT), indicating that DSBs were present. The presence of phosphorylated H2AX confirmed the creation of DSBs (Table 1). DNA-PKcs is Fig. 3. NER response to local Hoechst 33342 treatment + 405 nm recruited to DNA damage by Ku proteins (Downs and Jackson, irradiation. (A) XPC-GFP-expressing cells were irradiated through a 2004) and damage-induced autophosphorylation of DNA-PKcs filter with UV-C light (spots indicated by arrows), sensitized with is regulated by MDC1 (Lou et al., 2004) so we expected to find Hoechst 33342 and subsequently locally treated with 405 irradiation PKcs on local Hoechst 33342 + 405 nm damage. In contrast in the nucleus (lines indicated by arrowheads). Induction of CPDs is to its response to pulsed 800 nm irradiation, PKcs did not shown by the CPD antibody staining (right panel) both on UV-C and H+405 treated areas. XPC-GFP accumulated on both areas irradiated localize to Hoechst 33342-induced DNA damage in any of the through a filter with UV-C light (arrows) and irradiated with 405 nm irradiated cells above background levels of the in combination with Hoechst 33342 (arrowheads). (B) Treatment as immunohistochemical staining (Fig. 4C). Apparently the types in panel A, here cells were stained with an antibody that recognizes of lesions created with this method are not a good substrate for 6-4PPs (right panel). Surprisingly, no 6-4PP-staining can be detected PKcs. This indicates an activity of Ku70/Ku80 that is on laser-irradiated areas (lines indicated by arrowheads), while the independent of DNA-PKcs as was previously described for its UV-C treated areas show a clear induction (arrows). The bar graph proposed function at telomeres (Hsu et al., 2000). indicates fluorescence intensities of the nucleus (1), 405 nm Furthermore YFP-MDC1(BRCT) accumulation did not show combined with Hoechst 33342 treatment induced local damage (2) a focal pattern but rather was homogenously distributed within and UV-C induced local damage (3). (C) GFP-XPA accumulates to a the damaged area (Fig. 4A, right panel). Similarly Rad54-GFP low level on local damage induced by 405 nm laser irradiation in combination with Hoechst 33342 treatment (arrowhead) compared accumulated on damages induced by 405 nm in combination with local UV-C irradiated areas (arrow). The bar graph indicates with Hoechst 33342, albeit in low numbers (bar graph Fig. 4D), fluorescence intensities of the nucleus (1), 405 nm combined with but it did not appear in foci (Fig. 4D, right panel), not even after Hoechst 33342 treatment induced local damage (2) and UV-C 40 minutes (data not shown). Together with the absence of induced local damage (3). PKcs at irradiated areas this indicates that the combination of Hoechst 33342 sensitization and 405 nm light triggers a hitherto unknown response of DSB repair proteins. NER response to Hoechst 33342 + 405 nm damage induction NER and DSBs upon local UV-C irradiation The DNA binding agents Hoechst 33258 and 33342 are known To induce local UV damage, we installed a pulsed 2 mW 266 to induce DNA breaks when activated by UV-A irradiation nm laser on a confocal microscope adapted for UV-C (Lecoeur, 2002). Surprisingly, also the NER protein XPC-GFP, transmission with all-quartz optics. Local UV irradiation which detects 6-4PPs, and to a lesser extent CPDs, induced by through a micro-porous filter to inflict light-induced DNA UV-C (<300 nm), was targeted to 405 nm (UV-A)-irradiated damage is technically fairly easy, but includes a number of lines in Hoechst 33342-containing cells (Fig. 3A). In the drawbacks that are overcome with the use of a laser. First, absence of Hoechst 33342, DNA damage induction with a 405 unless a set-up is used where irradiation takes place on the nm laser required more than tenfold higher laser intensity (data microscope stage (Mone et al., 2004), irradiation through a not shown). This localization of the UV damage sensor XPC filter is unsuitable for the study of accumulation rates. Even prompted us to further analyze the types of DNA lesions with the use of the on-the-microscope-stage set-up, early or introduced by this procedure. XPC-GFP-expressing cells were quick assembly rates are hard to monitor because of the UV-C irradiated through a filter as an internal control for the relatively long irradiation times required (>12 seconds). Journal of Cell Science Laser-induced DNA damage 2735 UV-C light is known to directly induce helix-distorting lesions such as CPDs 6-4PPs but not SSBs or DSBs (Perdiz et al., 2000; Rodrigo et al., 2000). However, at high UV-C intensity positive TUNEL staining was found next to the accumulation of the NER factor XPA (Fig. 5A, arrowhead). In addition, the DSB factor Ku80-GFP accumulated in the irradiated area (footnote Table 1). At ~12-fold lower irradiation intensity, only the NER factors accumulated in the damaged region, indicating that NER-specific lesions were created both at high and at low intensities (Fig. 5A,B). Dose-dependency studies showed that up to 6 seconds irradiation with 12-fold attenuation induces accumulation of GFP-XPA but not of Ku80-GFP and that without attenuation 1-second irradiation was sufficient to induce DSBs (Table S1 in supplementary material). In the remaining experiments, the UV-C dose used was 0.5 seconds with 12-fold attenuation. After local irradiation with this dose, GFP-PCNA-expressing cells (Essers et al., 2005) were still able to go through mitosis (Fig. S2, and Movie 1 in supplementary material), suggesting that under the conditions used we did not trigger apoptosis. During S-phase, replication forks can stall when they encounter a UV-induced lesion. HR is suggested to be involved in resolving these stalled replication forks. Therefore we examined the response of Rad54 to UV-C laser irradiation at different stages of the cell-cycle in cells expressing both Rad54-GFP and mCherry-PCNA. In cells that showed a homogeneous mCherry-PCNA staining (G1 or G2 phases), no accumulation of Rad54-GFP at damaged areas was found (Fig. 5C). In cells with a focal mCherry-PCNA pattern, Rad54-GFP accumulated at irradiated areas (Fig. 5D), suggesting that HR is only activated by UV-C laser irradiation during replication. Fig. 4. DSB repair response to Hoechst 33342 + 405 nm damage. Accumulation kinetics with laser assisted DNA (A) YFP-MDC1(BRCT) expressing cells were incubated with damaging methods Hoechst 33342 and irradiated in an area of approximately 5 m We have measured the kinetic behavior of four DNA damage (white box) with 405 nm laser-light and the fluorescence repair proteins, XPC, XPA, MDC1 and Rad54, upon redistribution was followed in time. YFP-MDC1(BRCT) recruitment to the various local laser-damaged areas discussed accumulates in a non-focal/homogenous pattern at the damaged area above. To this end we monitored protein redistribution for up (right panel). (B) Ku-GFP expressing cells were incubated with Hoechst 33342 and irradiated with 405 nm light in a big (top cell) or to 20 minutes after local damage induction with either pulsed small (lower cell) area in the nucleus (white boxes). The 800 nm irradiation, 405 nm combined with Hoechst 33342 or accumulation of Ku-GFP was followed in time (right panel). 266 nm laser irradiation and compared fold increase of (C) PKcs does not accumulate at Hoechst 33342 + 405 nm treated fluorescence in the damaged area over time for these three sites (line in the nucleus indicated by arrowhead). The bright spots in damaging methods (Fig. 6A-D). the PKcs channel are described in Fig. 2A and are also found in XPC-GFP responded quickly to both the pulsed 800 nm cells that were not damaged. (D) Rad54-GFP expressing cells were and 266 nm irradiation methods but it accumulated slower at treated with Hoechst 33342 and 405 nm irradiation (white boxes) and Hoechst 33342 + 405 nm induced damage sites (Fig. 6A). fluorescence redistribution was followed in time (right panel). This unexpected behavior of XPC is most likely caused by an Rad54-GFP accumulates in very low numbers at locally damaged inhibitory effect of the presence of Hoechst on XPC mobility areas in a non-focal/homogenous pattern. The bar graph indicates the fluorescence intensity in the nucleus (1) and at the locally damaged (our unpublished work). XPC appeared to very transiently area (2). and frequently bind to Hoechst-stained DNA thus limiting the speed of its accumulation in the damaged area. GFP-XPA was not visibly retarded by Hoechst 33342 addition, but it accumulated to a much lesser extent than XPC- Second, irradiation through a filter induces damage in all cells GFP in areas exposed to either pulsed 800 nm irradiation or in the preparation simultaneously, making it very difficult to Hoechst combined with 405 nm irradiation. Both GFP-XPA monitor protein accumulations in multiple cells in one (Fig. 6B) and XPC-GFP (Fig. 6A) showed a stronger increase experiment. Laser irradiation provides much more flexibility, in fluorescence intensity with the 266 nm method than with the allowing local damage infliction at specific locations in other two, indicating that a UV-C laser can induce a high individual cells, e.g. specific sub-nuclear hetero- or concentration of lesions that are specifically repaired by NER euchromatic regions or even multiple irradiations in one cell or without creating DSBs at the same time (Tables 1 and 2). Note different doses in different cells in the same view, which is not that GFP-XPA took much longer to reach a plateau level in possible with filter irradiation. Journal of Cell Science 2736 Journal of Cell Science 120 (15) Mre11-Rad50-Nbs1 complex and chromatin (Lukas et al., 2004; Stucki et al., 2005). In agreement with its early association with damage sites, we found rapid accumulation of this protein at both pulsed 800 nm- and Hoechst + 405 nm- irradiated sites (Fig. 6C). Contrary to XPC, MDC1 accumulated faster in Hoechst-treated cells than in 800 nm- irradiated cells. Interestingly, Rad54-GFP displayed a delayed response to pulsed 800 nm damage, only visibly accumulating after 10 minutes (Fig. 6D). This is consistent with its proposed function later in the DSB repair process and suggests that the kinetics of HR are slower than that of NER of UV lesions (Essers et al., 2002a; Houtsmuller et al., 1999; Mone et al., 2004). It has been shown previously that Rad51, another HR factor, appears at local damage in a comparable timeframe (30 minutes) after DSB induction by a 532 nm laser and that it is still found at these sites after at least 24 hours (Kim et al., 2005). Rad54-GFP accumulated to a lesser extent but with faster kinetics in areas irradiated at 405 nm in Hoechst-treated cells than in areas irradiated by pulsed 800 nm. The combination of the homogeneous pattern of accumulation of both Rad54 and MDC1 and the absence of detectable PKcs accumulation suggests that the cellular response to 405 nm irradiation and Hoechst 33342 treatment is very different from the response to pulsed 800 nm irradiation. In addition, it suggests that different types of DNA damage are created with these methods and not just different amounts of the same type of damage. Discussion We have investigated the response of several DNA repair Fig. 5. UV-C laser irradiation. (A) GFP-XPA expressing cells were factors that are involved in either NER or DSB break repair, to irradiated with 266 nm either without (arrow) or with attenuation (arrowhead). GFP-XPA accumulates on both areas (green, left panel) different types of DNA damage induction (Tables 1 and 2). We whereas TUNEL (red, middle panel) only stains positive on the spot show that the NER factor XPC responds to many different that was created without attenuation. (B) GFP-XPA expressing cells types of lesions. This is illustrated, for example, by the strong were irradiated by attenuated UV-C laser light (arrow). Presence of response of XPC-GFP to pulsed 800 nm irradiation and 405 CPDs was shown by immunohistochemical staining with -CPD nm irradiation after Hoechst treatment, whereas GFP-XPA is (red, middle panel). (C) Cells that were irradiated in G1 or G2 phase recruited to irradiated areas to a much lesser extent. Upon 266 (homogeneous PCNA pattern, red, middle panel) show no nm irradiation this difference is much smaller. Interestingly, accumulation of Rad54-GFP (green, left panel) 2 hours after XPC-GFP seemed to be the only NER factor that accumulated irradiation (arrow). In cells that were irradiated in S phase (PCNA after irradiation with a 365 nm laser (Lan et al., 2004), pattern in foci, red, middle panel) Rad54-GFP (green, left panel) confirming that it binds to a wide range of DNA lesions and accumulates at locally irradiated areas within 1 hour after irradiation (arrow). not only to lesions that are repaired by NER. This is in accordance with previous in vitro DNA binding experiments showing low specificity of XPC for various aberrant DNA response to 266 nm irradiation than XPC-GFP (t values of structures (Sugasawa et al., 1998). In addition, live cell studies 1/2 ~140 and ~40 seconds, respectively). Two scenarios can using fluorescence recovery after photobleaching on XPC- explain this difference between XPA and XPC. (1) At GFP-expressing cells exposed to a variety of DNA damaging individual repair sites XPC is released before repair is agents known to induce lesions other than pyrimidine dimers, complete (Park and Choi, 2006; Riedl et al., 2003; You et al., showed participation of XPC-GFP similar to its behavior after 2003), whereas XPA remains bound for longer. A consequence UV-exposure (our unpublished work). of this difference in residence time is that XPC kinetics reach This observed affinity of XPC for a variety of DNA lesions equilibrium between binding and dissociation earlier than suggests the rapid formation of pre-repair complexes on DNA. XPA. (2) Alternatively, the association of XPA with locally Such a quick response may initiate rapid activation of cell- damaged areas is delayed because it depends on the presence cycle checkpoints after damage detection. The initial, weakly or enzymatic activity of an earlier factor (Mone et al., 2004; specific response is then followed by a more lesion-specific, Politi et al., 2005). but slower acting, damage verification step, which if positive, MDC1 has been found to interact with proteins of both the may, in its turn, activate a fully specific repair pathway required NHEJ and HR pathways (Bekker-Jensen et al., 2005; Lou et for the type of damage encountered. In addition, rapid al., 2004; Zhang et al., 2005) and is involved in early events in exchange of damage recognition proteins with more pathway- the DSB repair process, serving as an intermediary between the specific factors may ensure that a repair pathway can quickly Journal of Cell Science Laser-induced DNA damage 2737 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. become completely activated. Recently, such differential Many studies have been published in which DNA is sensitized dynamic interactions have been suggested to occur during prior to local irradiation. Sensitization of DNA can be transcription initiation (Hager et al., 2006; Metivier et al., accomplished by incorporation of a halogenated thymidine 2006). It was suggested that this prevents slowing down the analogue in combination with Hoechst (Limoli and Ward, 1993; entire transcription machinery due to too many non-productive Paull et al., 2000; Rogakou et al., 1999), by incorporation of long-lasting associations. A bipartite damage-recognition step halogenated Hoechst (Martin et al., 1994; Martin et al., 1990) or for NER has been suggested previously (Dip et al., 2004; of halogenated thymidine analogues alone (Lukas et al., 2003; Sugasawa et al., 2001) with quick binding of a low-specificity Tashiro et al., 2000). Halogenation is thought to be required for initiating factor (XPC) and subsequent lesion verification. Our DSB induction. However, Hoechst (either 33258 or 33342) by current data supports this model. itself can also sensitize DNA to UV-A irradiation resulting in DSB formation (Bradshaw et al., 2005; Celeste et al., 2003; Laser-assisted damaging techniques Kruhlak et al., 2006). Similarly, we have shown here that in the Formation of DSBs by a pulsed 800 nm laser has been reported absence of halogen intermediates, irradiation of Hoechst 33342- previously (König et al., 2001; Tirlapur and König, 2001) and sensitized cells at 405 nm induced DSBs, although it invokes a is thought to be caused by ablation of the DNA at the highly different response by Rad54 and PKcs, i.e. non-focal focused laser spot. In metaphase chromosomes this accumulation and absence at damaged sites, respectively, than multiphoton ablation introduces gaps of approximately 100 nm those induced by a pulsed 800 nm laser. Another remarkable corresponding to ~65 kb (König et al., 2001). Most likely such effect of 405 nm irradiation of Hoechst 33342-sensitized cells is gaps, i.e. DSBs, will be created in interphase chromosomes as the specific induction of CPDs but not 6-4PPs. well, explaining the accumulation of DSB repair proteins Photoisomerization of 6-4PPs results in the formation of the observed here. Recently, also the induction and repair of DSBs DewarPP, a photoproduct that is not recognized by the 6-4PP in living cultured cells has been described using this DNA antibody (Kobayashi et al., 2001). However, the optimum damage induction method (Mari et al., 2006). wavelength for photoisomerization is between 280 and 360 nm, A pulsed 800 nm laser beam has been shown to efficiently so 405 nm laser irradiation probably does not induce DewarPP induce CPDs (Meldrum et al., 2003) and here we show that formation. Instead, Hoechst binding induces local structural also 6-4PPs are efficiently formed with a pulsed 800 nm laser. changes in the DNA, which might not allow the bending angle The formation of these lesions, which are typically created by that is necessary for 6-4PP formation (Chen et al., 1993). We UV-C, is likely caused by three-photon absorption on the DNA, and others have noted that pre-sensitization of cells with Hoechst the effective wavelength being ~267 nm. 33343 induces a very broad spectrum of events associated with Journal of Cell Science 2738 Journal of Cell Science 120 (15) structural changes in the DNA conformation, ranging from of cellular repair mechanisms. The relative proportion of the chromosome decondensation (Turner and Denny, 1996) to induced damages, which determines the extent to which transcription inhibition (White et al., 2000). The aberrant different repair pathways become activated, is shown to differ responses shown here are: (1) absence of phosphorylated DNA- for the three studied methods. Proteins that respond to a variety PKcs from damaged areas, whereas DSBs are judged to have of lesions, such as XPC, will exhibit different kinetic behaviors formed by accumulation of Ku70-GFP; (2) reduced mobility of depending on the method used. In future studies, using more XPC; (3) homogenous accumulation of DSB repair proteins, than one source of DNA damage to study cellular responses, rather than the common focal pattern and (4) very rapid with accurate analysis of the types of lesions induced with accumulation of YFP-MDC1(BRCT) and Rad54-GFP these methods, will greatly help our understanding of DNA compared with the response to pulsed 800 nm irradiation. repair in vivo. Recently, also an aberrant accumulation of TRF2, a telomere binding protein, in response to local damage inflicted by pre- Materials and Methods sensitization with Hoechst combined with high intensity 800 nm Preparation and culture of cell lines XPC-GFP and GFP-XPA were expressed in the human cell lines XP4PA-SV and laser irradiation has been described, which has not been found XP2OS-SV, which are deficient in XPC and XPA, respectively (Politi et al., 2005; using many other local damage techniques (Williams et al., Rademakers et al., 2003). Rad54-GFP-expressing cell lines were created by stable 2007). We conclude that treatment with Hoechst 33343 as a expression of Rad54-GFP in CHO9 cells as described previously (Essers et al., 2002a). mCherry-PCNA was transfected into this cell line. The YFP-MDC1 sensitizer for DNA damage induction may have considerable (BRCT) cell line was created by stable expression of a construct encoding a YFP consequences for the cellular response. fusion to the BRCT domains of human MDC1 in CHO9 cells. This construct was Sensitization with halogenated nucleotides instead of shown to be functional as a marker for MDC1 localization (O’Driscoll et al., 2003). GFP-Ku80 was transfected into Ku-deficient XR-V15B cells (Mari et al., 2006). Hoechst prior to UV-A irradiation induces a response that is GFP-PCNA was expressed in CHO9 cells as described previously (Essers et al., much more similar to ionizing radiation and pulsed 800 nm 2005). All cell lines were cultured under standard conditions in DMEM-F10 irradiation as repair proteins accumulate in foci (Lukas et al., medium supplemented with 10% fetal calf serum and antibiotics at 37°C in 5% CO . 2003; Bekker-Jensen et al., 2006). One striking difference Local UV induction with UV-C lamps between pulsed 800 nm irradiation and UV-A irradiation of To induce local UV damage, cells were grown on coverslips, washed with PBS, halogenated thymidine-sensitized nuclei is the response of covered with a polycarbonate filter (5 m pore size; Millipore), irradiated with 100 NHEJ factors such as Ku80 and DNA-PKcs, which clearly J/m (overall dose) and incubated in standard growth medium for 30 minutes before accumulate in damaged areas created by the former but not by fixation or further treatment. the latter method. Probably, these methods induce a different Laser-induction of local damage spectrum of DNA lesions, for example blunt-ended DSBs A Coherent Mira modelocked Ti:Sapphire laser was used at 800 nm with a versus breaks with overhangs. Perhaps the relative pulselength of 200 fs and repetition rate of 76 MHz. Maximum output power on the cells for DNA damage induction was approximately 80 mW. concentration of these two types of DSBs determines the extent For the Hoechst + 405 nm treatment a 30 mW 405 nm diode laser supplied by to which NHEJ or HR becomes activated. Zeiss was used. Damage was induced at 60% of maximum power. We show that UV-C laser irradiation can induce pyrimidine For UV laser irradiation a 2 mW pulsed (7.8 kHz) diode pumped solid state laser dimers as well as DSBs, however, the latter only occurs after emitting at 266 nm (Rapp OptoElectronic, Hamburg GmbH) was connected to a Zeiss LSM 510 confocal microscope with an Axiovert 200 M housing adapted for high intensity irradiation. UV by all-quartz optics. A special adaptor (ZSI-A200, Rapp OptoElectronic) to fit in the aperture slider position of an Axiovert 200 microscope was developed by Specific DNA damage induction Rapp OptoElectronic to focus the laser on a sample. For local UV-C irradiation experiments cells were grown on 25 mm diameter quartz coverslips (010191T-AB, We show here that UV-C laser irradiation at the appropriate SPI supplies). intensity is the most specific method to induce 6-4PPs and CPDs. By contrast, induction of exclusively DSBs seems not Imaging of cells using confocal microscopy possible with currently existing laser-assisted damaging Cells expressing GFP-tagged repair factors were grown on coverslips and imaged at 37°C using a Zeiss confocal microscope setup (Zeiss LSM510). In the case of methods. This problem was overcome by a method specifically cells to be treated with a combination of Hoechst and 405 nm light, Hoechst 33342 inducing DSBs using a recombination reporter system was added to the medium (final concentration 0.5 g/ml) shortly before treatment. involving an HO or I-SceI endonuclease site adjacent to a Lac- Cells with an intermediate fluorescence level were selected to be treated with either 405 nm or 800 nm light. All treated cells were analyzed at the same magnification or Tet-operon repeat (Lisby et al., 2003; Miyazaki et al., 2004; and zoom factor using low laser power to minimize photobleaching during data Rodrigue et al., 2006). After induction of expression of the collection. The region to be damaged was always the same size and shape, and laser appropriate endonuclease, accumulation of repair proteins at treatment was done with calibrated lasers at the same laser output, to exclude the single DSB can be studied. This method has provided variations in dose. insight in the nature of repair foci, showing that multiple DSBs Immunofluorescence analysis can colocalize within one focus in yeast (Lisby et al., 2003). For immunohistochemical analysis, cells were washed with PBS and fixed for 15 Production of a known amount of well-specified DSBs will minutes in 2% paraformaldehyde in PBS 30-60 minutes after damage induction. become a valuable tool in the study of DSB repair, especially Next, the cells were washed with 3% BSA in PBS. In the case of antibodies directed against CPDs (TDM2) (Mori et al., 1991) or 6-4PPs (6-4-M-2) (Mori et al., 1991) since it has recently been effectively applied in mammalian cells were treated with 0.07 M NaOH in PBS for 5 minutes at room temperature to cells (Rodrigue et al., 2006). However, the study of denature the DNA. Next, the cells were washed three times with P-buffer (0.1% accumulation kinetics of DSB repair factors may be more Triton X-100 in PBS) and washed once using I-buffer (0.1% glycine, 1% BSA in PBS). Then, cells were incubated with primary antibodies (diluted in I-buffer) for complicated with this method because the timing of the activity 1 hour at 20°C for detection of protein epitopes or 12 hours at 4°C for detection of of restriction enzymes is difficult to control. DNA lesions. The rabbit anti-H2AX (Ser138) antibody was from Upstate Biotechnology (Charlottesville, VA, USA). After incubation, cells were washed three times using P-buffer, once using I-buffer, and incubated for 1 hour at 20°C Conclusion with secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 594 (or We have shown that most presently available and widely used multiple antibodies for double staining) diluted in I-buffer. Next, cells were washed laser-assisted DNA damaging methods induce a wide response three times using P-buffer, once with PBS and embedded in Vectashield (Vector Journal of Cell Science Laser-induced DNA damage 2739 Laboratory). The rabbit anti-PKcs antibody was a kind gift from D. Chen (Chan of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo. Mol. et al., 2002). The TUNEL staining method was acquired from Roche Applied Cell 10, 1163-1174. Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten, D., Hoeijmakers, J. Science, Penzberg Germany (Cat. No. 12156792910). PARP-1 accumulation was H. J. and Vermeulen, W. (1999). Action of DNA repair endonuclease ERCC1/XPF detected with anti-poly(ADP-ribose) polymerase-1 (human) polyclonal antibody in living cells. Science 284, 958-961. (ALX-210-895) from Alexis (Breda, The Netherlands). Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. 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Activation of multiple DNA repair pathways by sub-nuclear damage induction methods

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Abstract

Research Article 2731 Activation of multiple DNA repair pathways by sub- nuclear damage induction methods 1,2, 2, ,‡ 2,3 4 2,3 Christoffel Dinant *, Martijn de Jager * , Jeroen Essers , Wiggert A. van Cappellen , Roland Kanaar , 1,§ 2,§ Adriaan B. Houtsmuller and Wim Vermeulen 1 2 3 Department of Pathology, Josephine Nefkens Institute, Department of Cell Biology and Genetics, Department of Radiation Oncology and Department of Reproduction and Development, ErasmusMC, Rotterdam, The Netherlands *These authors contributed equally to this work Present address: Physics of Life Processes, Leiden Institute of Physics (LION), Leiden University, Leiden, The Netherlands Authors for correspondence (e-mails: [email protected]; [email protected]) Accepted 30 May 2007 Journal of Cell Science 120, 2731-2740 Published by The Company of Biologists 2007 doi:10.1242/jcs.004523 Summary Live cell studies of DNA repair mechanisms are greatly cellular response to presensitization by Hoechst 33342 and enhanced by new developments in real-time visualization subsequent 405 nm irradiation were aberrant from those of repair factors in living cells. Combined with recent to every other DNA damaging method described here or in advances in local sub-nuclear DNA damage induction the literature. Whereas, application of low-dose 266 nm procedures these methods have yielded detailed laser irradiation induced only UV-specific DNA photo- information on the dynamics of damage recognition and lesions allowing the study of the UV-C-induced DNA repair. Here we analyze and discuss the various types of damage response in a user-defined area in cultured cells. DNA damage induced in cells by three different local damage induction methods: pulsed 800 nm laser irradiation, Hoechst 33342 treatment combined with 405 Supplementary material available online at nm laser irradiation and UV-C (266 nm) laser irradiation. http://jcs.biologists.org/cgi/content/full/120/15/2731/DC1 A wide variety of damage was detected with the first two methods, including pyrimidine dimers and single- and Key words: Pyrimidine dimers, Local DNA damage induction, double-strand breaks. However, many aspects of the Double-strand breaks, Living cells, DNA repair kinetics Introduction al., 2001; Katsumi et al., 2001; Mone et al., 2001; Nelms et al., The mammalian genome is protected against the continuous 1998; Volker et al., 2001) to focusing laser beams inside living stress of both exogenous and endogenous DNA damaging cell nuclei (Essers et al., 2006; Lukas et al., 2005). agents by a number of DNA damage response mechanisms, The kinetics of nucleotide excision repair (NER) have been including different DNA repair pathways. Unresolved DNA determined previously by irradiation of cultured cells through lesions may introduce mutations, which can lead to cancer a polycarbonate filter with UV-C light, either prior to or after (Mitchell et al., 2003). In addition, unrepaired damages may mounting on the microscope stage, and subsequently result in disturbed transcription and replication, which measuring the accumulation of repair proteins (Hoogstraten et eventually causes cell death contributing to aging. The severe al., 2002; Mone et al., 2004; Politi et al., 2005; Zotter et al., clinical consequences associated with hereditary disorders that 2006). In addition, alternative methods have been developed harbor defects in DNA repair systems underscore the where DNA damage is introduced by focused laser beams, at importance of efficient DNA repair (Bootsma and user-defined regions within the nucleus (Cremer et al., 1980; Hoeijmakers, 1994; Hoeijmakers, 2001). Lan et al., 2004; Meldrum et al., 2003; Walter et al., 2003). Genetic and biochemical analysis of repair processes have This approach allows great flexibility not only with respect to culminated in detailed mechanistic insight into the distinct position, but also size and shape of the local damage induced DNA repair processes. To study the interaction of the different in individual cells. DNA repair processes with each other and with other cellular Tuned localized intense laser irradiation with 365 nm light processes such as transcription and replication, spatiotemporal causes different types of DNA lesions ranging from oxidized analysis of different DNA repair systems in intact living cells base damage, single-strand breaks (SSBs) and up to double- is required and has been used extensively with the aid of GFP- strand breaks (DSBs) (Lan et al., 2004). Another powerful tagged repair factors (Essers et al., 2002b; Hoogstraten et al., method uses pulsed near infrared laser (multiphoton) 2002; Houtsmuller et al., 1999; Rademakers et al., 2003). technology. In this case two or three lower energy photons are Recently, DNA repair research has been boosted substantially absorbed simultaneously resulting in twice or three times the by the development of several methods to locally inflict DNA energy deposition. Meldrum et al. (Meldrum et al., 2003) damage in cultured living cells, enabling the direct applied this procedure using a pulsed 750 nm laser (with an visualization of GFP-tagged repair factors accumulating at the effective wavelength of 250 nm) and showed that this method sub-nuclear region where the damage is caused. These methods is able to create UV-like DNA lesions in living cells as shown range from irradiating partially shielded cells (Kannouche et by in situ immunostaining using antibodies against cyclobutane Journal of Cell Science 2732 Journal of Cell Science 120 (15) Table 1. Induced damages and protein accumulations: DSB and SSB repair Treatment TUNEL H2AX PKcs MDC1 Rad54 Ku70 PARP-1 Pulsed 800nm laser + + + + + + + Hoechst + 405 nm laser + + – +* +* + + † ‡ § § † UV-C – – –– – – – *DSB repair proteins that do not accumulate in foci but in a homogenous pattern; UV-C irradiation without attenuation resulted in positive TUNEL staining ‡ § and Ku-GFP accumulation; at higher UV-C doses H2AX accumulation can be found; accumulation of DSB repair proteins on UV-C damage is dependent on ongoing replication. Table 2. Induced damages and protein accumulations: pyrimidine dimers (CPDs). Recently, it has been shown that nucleotide excision repair (NER) with a pulsed near infrared laser DSBs are created as well Treatment CPD 6-4PP XPC XPA (Mari et al., 2006), indicating the broad spectrum of DNA lesions induced with this procedure. Pulsed 800 nm laser + + + + Hoechst+405 nm laser + – + + More indirect methods rely on local relatively low energy UV-C + + + + UV-A irradiation. These methods require cells to be pretreated with halogenated thymidine analogs such as BrdU or IdU, which are incorporated into DNA, and induce SSBs and DSBs when the cells are exposed to UV-A (Lukas et al., 2003; Tashiro et al., 2000). A variant of this method employs DNA- Response of the NER machinery to pulsed 800 nm binding dyes such as Hoechst either in combination with irradiation (Rogakou et al., 1999; Walter et al., 2003), or without To investigate the types of DNA damage created by pulsed near thymidine analogs (Bradshaw et al., 2005). Although a number infrared (NIR) laser irradiation, cells were subjected to high of these in situ local damage-inducing systems have been intensity 800 nm laser pulses. To provide an internal control applied to study DNA damage response mechanisms the for the immunofluorescent detection of pyrimidine dimers, we spectrum of DNA lesion induced by these procedures has not irradiated XPC-GFP expressing cells with UV-C light through been analyzed in great detail. a filter before irradiation with a NIR laser. Pulsed 800 nm laser We have systematically analyzed and compared three irradiation resulted in the formation of CPDs (Fig. 1A), as different procedures to locally inflict DNA damage in cultured reported previously (Meldrum et al., 2003). In addition to cells. We show that pulsed 800 nm irradiation introduces a CPDs, also 6-4PPs were formed (Fig. 1B; arrowheads). XPC- broad variety of DNA lesions at which proteins involved in GFP (Politi et al., 2005) accumulated in areas irradiated with different pathways accumulate. The combination of Hoechst a UV lamp through a micro-porous filter as well as areas 33342 incorporation and 405 nm irradiation induced a cellular irradiated with a pulsed 800 nm laser (Fig. 1A,B). GFP-XPA response that differed strongly from the response to other (Rademakers et al., 2003) also accumulated with both methods, damaging methods. In addition, we have developed a but there was a much stronger response to UV lamp irradiation microscope setting using focused UV-C (266 nm) laser than to pulsed 800 nm irradiation (Fig. 1C). irradiation, which induces predominantly UV-C-specific photolesions such as cyclobutane pyrimidine dimers (CPD) Response of the DSB repair machinery to pulsed and 6-4 photoproducts (6-4PP). 800 nm irradiation To determine whether DSBs are induced by a pulsed 800 nm Results laser we stained locally irradiated nuclei of XPC-deficient Experimental setup fibroblasts (XP4PA) expressing XPC-GFP (Politi et al., 2005) We have investigated local DNA damage induction in cultured with an antibody against phosphorylated DNA-PKcs (PKcs). living cells with confocal microscopy using lasers of different DNA-PKcs is the catalytic subunit of the DNA-dependent wavelengths: 800 nm, 405 nm and 266 nm. The types of protein kinase (DNA-PK), which is autophosphorylated in damages created with these methods and the assembly of response to ionizing radiation (Chan et al., 2002). The presence different repair proteins after local irradiation were first of PKcs suggested the formation of DSBs by a pulsed 800 analyzed using immunocytological procedures directed against nm laser (Fig. 2A). In addition, H2AX (Fig. 2B) and Ku80- lesions (CPD, 6-4PP and TUNEL) or the consequences of GFP (Mari et al., 2006) were also found at these sites, lesions (accumulation of phosphorylated H2AX, indicative of the presence of DSBs. Under similar conditions phosphorylated DNA-PKcs, PARP-1) as well as protein-GFP local UV-C irradiation through pores in a filter failed to induce fusions. The results of these studies are summarized in Tables DSBs as indicated by the absence of PKcs positive signal 1 and 2. In addition, the kinetics of protein interaction with (Table 1) and H2AX staining (Fig. 2B). DNA damage complexes were analyzed in living cells Rad54 is implicated in multiple steps of DSB repair through expressing fluorescently tagged repair factors involved in both homologous recombination (HR). Previous research has shown early and late steps of the reaction of both nucleotide excision that in response to DSB induction by ionizing radiation HR repair (involving XPC and XPA proteins) and DSB repair proteins accumulate in nuclear foci (Essers et al., 2002b; Rouse (MDC1and Rad54 proteins). All cell lines expressing GFP- or and Jackson, 2002; van Veelen et al., 2005a; van Veelen et al., YFP-tagged proteins have previously been characterized and 2005b). Accordingly, Rad54-GFP accumulated in a focal pattern published (see Materials and Methods section and references at the damaged area (Fig. 2C, right panel), similar to what has therein). been described for multiple HR proteins after DNA damage Journal of Cell Science Laser-induced DNA damage 2733 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 Fig. 2. The DSB repair response to local pulsed 800 nm laser intensities of the nucleus (1), pulsed 800 nm induced local damage irradiation. (A) XPC-GFP-expressing cells were treated with pulsed (2) and UV-C induced local damage (3). (C) GFP-XPA accumulates 800 nm irradiation and presence of DSBs is shown by to a limited extent on pulsed 800 nm induced damaged areas immunohistochemical staining with a PKcs antibody (lines in right (arrowhead) compared to UV-C irradiated areas (arrow). The bar panel indicated by arrowheads). The bright spots outside the damaged graph indicates fluorescence intensities of the nucleus (1), pulsed area in the right panel are nucleolar structures of unknown origin and 800 nm induced local damage (2) and UV-C induced local damage it is unknown if they exist in a living cell as well. (B) XPC-GFP- (3). 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 induction (Bekker-Jensen et al., 2006). Whereas HR is thought presence of DSBs (right panel, arrowheads). No accumulation of to be predominantly active during the S and G2 phases of the H2AX is found on UV-C irradiated spots (arrows). Earlier it was shown that phosphorylation of H2AX takes place after UV-C cell-cycle, we found accumulation of Rad54-GFP in virtually all irradiation (Marti et al., 2006; O’Driscoll et al., 2003) and we have cells. Similarly, Rad51 was found to accumulate at locally found this as well in other experiments (data not shown). It is possible damaged areas regardless of cell-cycle phase (Kim et al., 2005). that in this case the specific immunohistochemical staining of H2AX These observations suggest that part of the HR machinery is at UV-C damage was not strong enough to be detected over loaded onto DSBs in G1. However, this recruitment might not background signals. (C) Rad54-GFP expressing cells were irradiated reflect ongoing repair. Interestingly, the BRCT domain of MDC1 2 in an area of approximately 5 m with pulsed 800 nm light and the tagged with YFP [YFP-MDC1(BRCT)] also accumulated on redistribution of fluorescence was studied in time. The boxed area is pulsed 800 nm laser-induced damage, but with faster kinetics two times enlarged in the left bottom of both panels. Rad54-GFP and in much bigger foci than Rad54 (Fig. 2D, right panel, versus accumulates in small foci at the damaged area. (D) YFP- Fig. 2C, right panel). These large foci are likely indicative of MDC1(BRCT) expressing cells were irradiated in a rectangular line through the nucleus and fluorescence redistribution was followed in interaction between MDC1 and H2AX (Bekker-Jensen et al., time. YFP-MDC1(BRCT) accumulates in large foci at the damaged 2006). area (boxed area, left panel). To determine a dose of pulsed 800 nm radiation with which one specific repair pathway was induced and not another, we lowered the laser intensity. At slightly lower doses than used indicates that under the conditions used we did not observe above, both GFP-XPA (NER) and Rad54-GFP (HR) remained preferential formation of one type of lesion over the other by undetectable in the irradiated areas (data not shown). This changing the applied dose. Journal of Cell Science 2734 Journal of Cell Science 120 (15) pyrimidine dimer antibody staining before addition of Hoechst. After Hoechst 33342 treatment, a rectangular area of the nucleus of these cells was exposed to 405 nm irradiation. This resulted in abundant CPD formation (Fig. 3A) identical to pulsed 800 nm-induced damage (Fig. 1C). Remarkably, no 6- 4PPs were found (Fig. 3B). Apparently this method specifically induced minor helix distorting lesions such as CPDs but not the more severely helix distorting 6-4PPs. Similar to its response to pulsed 800 nm irradiation, XPC-GFP responded very strongly to these damages (Fig. 3A,B) but GFP-XPA accumulation was much less intense on Hoechst + 405 nm-irradiated areas than on UV lamp-irradiated areas (Fig. 3C). This suggests that XPC responds to a wider variety of lesions than only those typically repaired by NER. Response of the DSB repair machinery to Hoechst 33342 + 405 nm damage induction In Hoechst 33342-sensitized CHO9 cells locally irradiated at 405 nm, YFP-MDC1(BRCT) as well as the non-homologous end-joining (NHEJ)-specific Ku80-GFP quickly accumulated in the irradiated areas in very high numbers (Fig. 4A,B). See also Fig. S1 in supplementary material for colocalization of XPC-mCherry and YFP-MDC1(BRCT), indicating that DSBs were present. The presence of phosphorylated H2AX confirmed the creation of DSBs (Table 1). DNA-PKcs is Fig. 3. NER response to local Hoechst 33342 treatment + 405 nm recruited to DNA damage by Ku proteins (Downs and Jackson, irradiation. (A) XPC-GFP-expressing cells were irradiated through a 2004) and damage-induced autophosphorylation of DNA-PKcs filter with UV-C light (spots indicated by arrows), sensitized with is regulated by MDC1 (Lou et al., 2004) so we expected to find Hoechst 33342 and subsequently locally treated with 405 irradiation PKcs on local Hoechst 33342 + 405 nm damage. In contrast in the nucleus (lines indicated by arrowheads). Induction of CPDs is to its response to pulsed 800 nm irradiation, PKcs did not shown by the CPD antibody staining (right panel) both on UV-C and H+405 treated areas. XPC-GFP accumulated on both areas irradiated localize to Hoechst 33342-induced DNA damage in any of the through a filter with UV-C light (arrows) and irradiated with 405 nm irradiated cells above background levels of the in combination with Hoechst 33342 (arrowheads). (B) Treatment as immunohistochemical staining (Fig. 4C). Apparently the types in panel A, here cells were stained with an antibody that recognizes of lesions created with this method are not a good substrate for 6-4PPs (right panel). Surprisingly, no 6-4PP-staining can be detected PKcs. This indicates an activity of Ku70/Ku80 that is on laser-irradiated areas (lines indicated by arrowheads), while the independent of DNA-PKcs as was previously described for its UV-C treated areas show a clear induction (arrows). The bar graph proposed function at telomeres (Hsu et al., 2000). indicates fluorescence intensities of the nucleus (1), 405 nm Furthermore YFP-MDC1(BRCT) accumulation did not show combined with Hoechst 33342 treatment induced local damage (2) a focal pattern but rather was homogenously distributed within and UV-C induced local damage (3). (C) GFP-XPA accumulates to a the damaged area (Fig. 4A, right panel). Similarly Rad54-GFP low level on local damage induced by 405 nm laser irradiation in combination with Hoechst 33342 treatment (arrowhead) compared accumulated on damages induced by 405 nm in combination with local UV-C irradiated areas (arrow). The bar graph indicates with Hoechst 33342, albeit in low numbers (bar graph Fig. 4D), fluorescence intensities of the nucleus (1), 405 nm combined with but it did not appear in foci (Fig. 4D, right panel), not even after Hoechst 33342 treatment induced local damage (2) and UV-C 40 minutes (data not shown). Together with the absence of induced local damage (3). PKcs at irradiated areas this indicates that the combination of Hoechst 33342 sensitization and 405 nm light triggers a hitherto unknown response of DSB repair proteins. NER response to Hoechst 33342 + 405 nm damage induction NER and DSBs upon local UV-C irradiation The DNA binding agents Hoechst 33258 and 33342 are known To induce local UV damage, we installed a pulsed 2 mW 266 to induce DNA breaks when activated by UV-A irradiation nm laser on a confocal microscope adapted for UV-C (Lecoeur, 2002). Surprisingly, also the NER protein XPC-GFP, transmission with all-quartz optics. Local UV irradiation which detects 6-4PPs, and to a lesser extent CPDs, induced by through a micro-porous filter to inflict light-induced DNA UV-C (<300 nm), was targeted to 405 nm (UV-A)-irradiated damage is technically fairly easy, but includes a number of lines in Hoechst 33342-containing cells (Fig. 3A). In the drawbacks that are overcome with the use of a laser. First, absence of Hoechst 33342, DNA damage induction with a 405 unless a set-up is used where irradiation takes place on the nm laser required more than tenfold higher laser intensity (data microscope stage (Mone et al., 2004), irradiation through a not shown). This localization of the UV damage sensor XPC filter is unsuitable for the study of accumulation rates. Even prompted us to further analyze the types of DNA lesions with the use of the on-the-microscope-stage set-up, early or introduced by this procedure. XPC-GFP-expressing cells were quick assembly rates are hard to monitor because of the UV-C irradiated through a filter as an internal control for the relatively long irradiation times required (>12 seconds). Journal of Cell Science Laser-induced DNA damage 2735 UV-C light is known to directly induce helix-distorting lesions such as CPDs 6-4PPs but not SSBs or DSBs (Perdiz et al., 2000; Rodrigo et al., 2000). However, at high UV-C intensity positive TUNEL staining was found next to the accumulation of the NER factor XPA (Fig. 5A, arrowhead). In addition, the DSB factor Ku80-GFP accumulated in the irradiated area (footnote Table 1). At ~12-fold lower irradiation intensity, only the NER factors accumulated in the damaged region, indicating that NER-specific lesions were created both at high and at low intensities (Fig. 5A,B). Dose-dependency studies showed that up to 6 seconds irradiation with 12-fold attenuation induces accumulation of GFP-XPA but not of Ku80-GFP and that without attenuation 1-second irradiation was sufficient to induce DSBs (Table S1 in supplementary material). In the remaining experiments, the UV-C dose used was 0.5 seconds with 12-fold attenuation. After local irradiation with this dose, GFP-PCNA-expressing cells (Essers et al., 2005) were still able to go through mitosis (Fig. S2, and Movie 1 in supplementary material), suggesting that under the conditions used we did not trigger apoptosis. During S-phase, replication forks can stall when they encounter a UV-induced lesion. HR is suggested to be involved in resolving these stalled replication forks. Therefore we examined the response of Rad54 to UV-C laser irradiation at different stages of the cell-cycle in cells expressing both Rad54-GFP and mCherry-PCNA. In cells that showed a homogeneous mCherry-PCNA staining (G1 or G2 phases), no accumulation of Rad54-GFP at damaged areas was found (Fig. 5C). In cells with a focal mCherry-PCNA pattern, Rad54-GFP accumulated at irradiated areas (Fig. 5D), suggesting that HR is only activated by UV-C laser irradiation during replication. Fig. 4. DSB repair response to Hoechst 33342 + 405 nm damage. Accumulation kinetics with laser assisted DNA (A) YFP-MDC1(BRCT) expressing cells were incubated with damaging methods Hoechst 33342 and irradiated in an area of approximately 5 m We have measured the kinetic behavior of four DNA damage (white box) with 405 nm laser-light and the fluorescence repair proteins, XPC, XPA, MDC1 and Rad54, upon redistribution was followed in time. YFP-MDC1(BRCT) recruitment to the various local laser-damaged areas discussed accumulates in a non-focal/homogenous pattern at the damaged area above. To this end we monitored protein redistribution for up (right panel). (B) Ku-GFP expressing cells were incubated with Hoechst 33342 and irradiated with 405 nm light in a big (top cell) or to 20 minutes after local damage induction with either pulsed small (lower cell) area in the nucleus (white boxes). The 800 nm irradiation, 405 nm combined with Hoechst 33342 or accumulation of Ku-GFP was followed in time (right panel). 266 nm laser irradiation and compared fold increase of (C) PKcs does not accumulate at Hoechst 33342 + 405 nm treated fluorescence in the damaged area over time for these three sites (line in the nucleus indicated by arrowhead). The bright spots in damaging methods (Fig. 6A-D). the PKcs channel are described in Fig. 2A and are also found in XPC-GFP responded quickly to both the pulsed 800 nm cells that were not damaged. (D) Rad54-GFP expressing cells were and 266 nm irradiation methods but it accumulated slower at treated with Hoechst 33342 and 405 nm irradiation (white boxes) and Hoechst 33342 + 405 nm induced damage sites (Fig. 6A). fluorescence redistribution was followed in time (right panel). This unexpected behavior of XPC is most likely caused by an Rad54-GFP accumulates in very low numbers at locally damaged inhibitory effect of the presence of Hoechst on XPC mobility areas in a non-focal/homogenous pattern. The bar graph indicates the fluorescence intensity in the nucleus (1) and at the locally damaged (our unpublished work). XPC appeared to very transiently area (2). and frequently bind to Hoechst-stained DNA thus limiting the speed of its accumulation in the damaged area. GFP-XPA was not visibly retarded by Hoechst 33342 addition, but it accumulated to a much lesser extent than XPC- Second, irradiation through a filter induces damage in all cells GFP in areas exposed to either pulsed 800 nm irradiation or in the preparation simultaneously, making it very difficult to Hoechst combined with 405 nm irradiation. Both GFP-XPA monitor protein accumulations in multiple cells in one (Fig. 6B) and XPC-GFP (Fig. 6A) showed a stronger increase experiment. Laser irradiation provides much more flexibility, in fluorescence intensity with the 266 nm method than with the allowing local damage infliction at specific locations in other two, indicating that a UV-C laser can induce a high individual cells, e.g. specific sub-nuclear hetero- or concentration of lesions that are specifically repaired by NER euchromatic regions or even multiple irradiations in one cell or without creating DSBs at the same time (Tables 1 and 2). Note different doses in different cells in the same view, which is not that GFP-XPA took much longer to reach a plateau level in possible with filter irradiation. Journal of Cell Science 2736 Journal of Cell Science 120 (15) Mre11-Rad50-Nbs1 complex and chromatin (Lukas et al., 2004; Stucki et al., 2005). In agreement with its early association with damage sites, we found rapid accumulation of this protein at both pulsed 800 nm- and Hoechst + 405 nm- irradiated sites (Fig. 6C). Contrary to XPC, MDC1 accumulated faster in Hoechst-treated cells than in 800 nm- irradiated cells. Interestingly, Rad54-GFP displayed a delayed response to pulsed 800 nm damage, only visibly accumulating after 10 minutes (Fig. 6D). This is consistent with its proposed function later in the DSB repair process and suggests that the kinetics of HR are slower than that of NER of UV lesions (Essers et al., 2002a; Houtsmuller et al., 1999; Mone et al., 2004). It has been shown previously that Rad51, another HR factor, appears at local damage in a comparable timeframe (30 minutes) after DSB induction by a 532 nm laser and that it is still found at these sites after at least 24 hours (Kim et al., 2005). Rad54-GFP accumulated to a lesser extent but with faster kinetics in areas irradiated at 405 nm in Hoechst-treated cells than in areas irradiated by pulsed 800 nm. The combination of the homogeneous pattern of accumulation of both Rad54 and MDC1 and the absence of detectable PKcs accumulation suggests that the cellular response to 405 nm irradiation and Hoechst 33342 treatment is very different from the response to pulsed 800 nm irradiation. In addition, it suggests that different types of DNA damage are created with these methods and not just different amounts of the same type of damage. Discussion We have investigated the response of several DNA repair Fig. 5. UV-C laser irradiation. (A) GFP-XPA expressing cells were factors that are involved in either NER or DSB break repair, to irradiated with 266 nm either without (arrow) or with attenuation (arrowhead). GFP-XPA accumulates on both areas (green, left panel) different types of DNA damage induction (Tables 1 and 2). We whereas TUNEL (red, middle panel) only stains positive on the spot show that the NER factor XPC responds to many different that was created without attenuation. (B) GFP-XPA expressing cells types of lesions. This is illustrated, for example, by the strong were irradiated by attenuated UV-C laser light (arrow). Presence of response of XPC-GFP to pulsed 800 nm irradiation and 405 CPDs was shown by immunohistochemical staining with -CPD nm irradiation after Hoechst treatment, whereas GFP-XPA is (red, middle panel). (C) Cells that were irradiated in G1 or G2 phase recruited to irradiated areas to a much lesser extent. Upon 266 (homogeneous PCNA pattern, red, middle panel) show no nm irradiation this difference is much smaller. Interestingly, accumulation of Rad54-GFP (green, left panel) 2 hours after XPC-GFP seemed to be the only NER factor that accumulated irradiation (arrow). In cells that were irradiated in S phase (PCNA after irradiation with a 365 nm laser (Lan et al., 2004), pattern in foci, red, middle panel) Rad54-GFP (green, left panel) confirming that it binds to a wide range of DNA lesions and accumulates at locally irradiated areas within 1 hour after irradiation (arrow). not only to lesions that are repaired by NER. This is in accordance with previous in vitro DNA binding experiments showing low specificity of XPC for various aberrant DNA response to 266 nm irradiation than XPC-GFP (t values of structures (Sugasawa et al., 1998). In addition, live cell studies 1/2 ~140 and ~40 seconds, respectively). Two scenarios can using fluorescence recovery after photobleaching on XPC- explain this difference between XPA and XPC. (1) At GFP-expressing cells exposed to a variety of DNA damaging individual repair sites XPC is released before repair is agents known to induce lesions other than pyrimidine dimers, complete (Park and Choi, 2006; Riedl et al., 2003; You et al., showed participation of XPC-GFP similar to its behavior after 2003), whereas XPA remains bound for longer. A consequence UV-exposure (our unpublished work). of this difference in residence time is that XPC kinetics reach This observed affinity of XPC for a variety of DNA lesions equilibrium between binding and dissociation earlier than suggests the rapid formation of pre-repair complexes on DNA. XPA. (2) Alternatively, the association of XPA with locally Such a quick response may initiate rapid activation of cell- damaged areas is delayed because it depends on the presence cycle checkpoints after damage detection. The initial, weakly or enzymatic activity of an earlier factor (Mone et al., 2004; specific response is then followed by a more lesion-specific, Politi et al., 2005). but slower acting, damage verification step, which if positive, MDC1 has been found to interact with proteins of both the may, in its turn, activate a fully specific repair pathway required NHEJ and HR pathways (Bekker-Jensen et al., 2005; Lou et for the type of damage encountered. In addition, rapid al., 2004; Zhang et al., 2005) and is involved in early events in exchange of damage recognition proteins with more pathway- the DSB repair process, serving as an intermediary between the specific factors may ensure that a repair pathway can quickly Journal of Cell Science Laser-induced DNA damage 2737 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. become completely activated. Recently, such differential Many studies have been published in which DNA is sensitized dynamic interactions have been suggested to occur during prior to local irradiation. Sensitization of DNA can be transcription initiation (Hager et al., 2006; Metivier et al., accomplished by incorporation of a halogenated thymidine 2006). It was suggested that this prevents slowing down the analogue in combination with Hoechst (Limoli and Ward, 1993; entire transcription machinery due to too many non-productive Paull et al., 2000; Rogakou et al., 1999), by incorporation of long-lasting associations. A bipartite damage-recognition step halogenated Hoechst (Martin et al., 1994; Martin et al., 1990) or for NER has been suggested previously (Dip et al., 2004; of halogenated thymidine analogues alone (Lukas et al., 2003; Sugasawa et al., 2001) with quick binding of a low-specificity Tashiro et al., 2000). Halogenation is thought to be required for initiating factor (XPC) and subsequent lesion verification. Our DSB induction. However, Hoechst (either 33258 or 33342) by current data supports this model. itself can also sensitize DNA to UV-A irradiation resulting in DSB formation (Bradshaw et al., 2005; Celeste et al., 2003; Laser-assisted damaging techniques Kruhlak et al., 2006). Similarly, we have shown here that in the Formation of DSBs by a pulsed 800 nm laser has been reported absence of halogen intermediates, irradiation of Hoechst 33342- previously (König et al., 2001; Tirlapur and König, 2001) and sensitized cells at 405 nm induced DSBs, although it invokes a is thought to be caused by ablation of the DNA at the highly different response by Rad54 and PKcs, i.e. non-focal focused laser spot. In metaphase chromosomes this accumulation and absence at damaged sites, respectively, than multiphoton ablation introduces gaps of approximately 100 nm those induced by a pulsed 800 nm laser. Another remarkable corresponding to ~65 kb (König et al., 2001). Most likely such effect of 405 nm irradiation of Hoechst 33342-sensitized cells is gaps, i.e. DSBs, will be created in interphase chromosomes as the specific induction of CPDs but not 6-4PPs. well, explaining the accumulation of DSB repair proteins Photoisomerization of 6-4PPs results in the formation of the observed here. Recently, also the induction and repair of DSBs DewarPP, a photoproduct that is not recognized by the 6-4PP in living cultured cells has been described using this DNA antibody (Kobayashi et al., 2001). However, the optimum damage induction method (Mari et al., 2006). wavelength for photoisomerization is between 280 and 360 nm, A pulsed 800 nm laser beam has been shown to efficiently so 405 nm laser irradiation probably does not induce DewarPP induce CPDs (Meldrum et al., 2003) and here we show that formation. Instead, Hoechst binding induces local structural also 6-4PPs are efficiently formed with a pulsed 800 nm laser. changes in the DNA, which might not allow the bending angle The formation of these lesions, which are typically created by that is necessary for 6-4PP formation (Chen et al., 1993). We UV-C, is likely caused by three-photon absorption on the DNA, and others have noted that pre-sensitization of cells with Hoechst the effective wavelength being ~267 nm. 33343 induces a very broad spectrum of events associated with Journal of Cell Science 2738 Journal of Cell Science 120 (15) structural changes in the DNA conformation, ranging from of cellular repair mechanisms. The relative proportion of the chromosome decondensation (Turner and Denny, 1996) to induced damages, which determines the extent to which transcription inhibition (White et al., 2000). The aberrant different repair pathways become activated, is shown to differ responses shown here are: (1) absence of phosphorylated DNA- for the three studied methods. Proteins that respond to a variety PKcs from damaged areas, whereas DSBs are judged to have of lesions, such as XPC, will exhibit different kinetic behaviors formed by accumulation of Ku70-GFP; (2) reduced mobility of depending on the method used. In future studies, using more XPC; (3) homogenous accumulation of DSB repair proteins, than one source of DNA damage to study cellular responses, rather than the common focal pattern and (4) very rapid with accurate analysis of the types of lesions induced with accumulation of YFP-MDC1(BRCT) and Rad54-GFP these methods, will greatly help our understanding of DNA compared with the response to pulsed 800 nm irradiation. repair in vivo. Recently, also an aberrant accumulation of TRF2, a telomere binding protein, in response to local damage inflicted by pre- Materials and Methods sensitization with Hoechst combined with high intensity 800 nm Preparation and culture of cell lines XPC-GFP and GFP-XPA were expressed in the human cell lines XP4PA-SV and laser irradiation has been described, which has not been found XP2OS-SV, which are deficient in XPC and XPA, respectively (Politi et al., 2005; using many other local damage techniques (Williams et al., Rademakers et al., 2003). Rad54-GFP-expressing cell lines were created by stable 2007). We conclude that treatment with Hoechst 33343 as a expression of Rad54-GFP in CHO9 cells as described previously (Essers et al., 2002a). mCherry-PCNA was transfected into this cell line. The YFP-MDC1 sensitizer for DNA damage induction may have considerable (BRCT) cell line was created by stable expression of a construct encoding a YFP consequences for the cellular response. fusion to the BRCT domains of human MDC1 in CHO9 cells. This construct was Sensitization with halogenated nucleotides instead of shown to be functional as a marker for MDC1 localization (O’Driscoll et al., 2003). GFP-Ku80 was transfected into Ku-deficient XR-V15B cells (Mari et al., 2006). Hoechst prior to UV-A irradiation induces a response that is GFP-PCNA was expressed in CHO9 cells as described previously (Essers et al., much more similar to ionizing radiation and pulsed 800 nm 2005). All cell lines were cultured under standard conditions in DMEM-F10 irradiation as repair proteins accumulate in foci (Lukas et al., medium supplemented with 10% fetal calf serum and antibiotics at 37°C in 5% CO . 2003; Bekker-Jensen et al., 2006). One striking difference Local UV induction with UV-C lamps between pulsed 800 nm irradiation and UV-A irradiation of To induce local UV damage, cells were grown on coverslips, washed with PBS, halogenated thymidine-sensitized nuclei is the response of covered with a polycarbonate filter (5 m pore size; Millipore), irradiated with 100 NHEJ factors such as Ku80 and DNA-PKcs, which clearly J/m (overall dose) and incubated in standard growth medium for 30 minutes before accumulate in damaged areas created by the former but not by fixation or further treatment. the latter method. Probably, these methods induce a different Laser-induction of local damage spectrum of DNA lesions, for example blunt-ended DSBs A Coherent Mira modelocked Ti:Sapphire laser was used at 800 nm with a versus breaks with overhangs. Perhaps the relative pulselength of 200 fs and repetition rate of 76 MHz. Maximum output power on the cells for DNA damage induction was approximately 80 mW. concentration of these two types of DSBs determines the extent For the Hoechst + 405 nm treatment a 30 mW 405 nm diode laser supplied by to which NHEJ or HR becomes activated. Zeiss was used. Damage was induced at 60% of maximum power. We show that UV-C laser irradiation can induce pyrimidine For UV laser irradiation a 2 mW pulsed (7.8 kHz) diode pumped solid state laser dimers as well as DSBs, however, the latter only occurs after emitting at 266 nm (Rapp OptoElectronic, Hamburg GmbH) was connected to a Zeiss LSM 510 confocal microscope with an Axiovert 200 M housing adapted for high intensity irradiation. UV by all-quartz optics. A special adaptor (ZSI-A200, Rapp OptoElectronic) to fit in the aperture slider position of an Axiovert 200 microscope was developed by Specific DNA damage induction Rapp OptoElectronic to focus the laser on a sample. For local UV-C irradiation experiments cells were grown on 25 mm diameter quartz coverslips (010191T-AB, We show here that UV-C laser irradiation at the appropriate SPI supplies). intensity is the most specific method to induce 6-4PPs and CPDs. By contrast, induction of exclusively DSBs seems not Imaging of cells using confocal microscopy possible with currently existing laser-assisted damaging Cells expressing GFP-tagged repair factors were grown on coverslips and imaged at 37°C using a Zeiss confocal microscope setup (Zeiss LSM510). In the case of methods. This problem was overcome by a method specifically cells to be treated with a combination of Hoechst and 405 nm light, Hoechst 33342 inducing DSBs using a recombination reporter system was added to the medium (final concentration 0.5 g/ml) shortly before treatment. involving an HO or I-SceI endonuclease site adjacent to a Lac- Cells with an intermediate fluorescence level were selected to be treated with either 405 nm or 800 nm light. All treated cells were analyzed at the same magnification or Tet-operon repeat (Lisby et al., 2003; Miyazaki et al., 2004; and zoom factor using low laser power to minimize photobleaching during data Rodrigue et al., 2006). After induction of expression of the collection. The region to be damaged was always the same size and shape, and laser appropriate endonuclease, accumulation of repair proteins at treatment was done with calibrated lasers at the same laser output, to exclude the single DSB can be studied. This method has provided variations in dose. insight in the nature of repair foci, showing that multiple DSBs Immunofluorescence analysis can colocalize within one focus in yeast (Lisby et al., 2003). For immunohistochemical analysis, cells were washed with PBS and fixed for 15 Production of a known amount of well-specified DSBs will minutes in 2% paraformaldehyde in PBS 30-60 minutes after damage induction. become a valuable tool in the study of DSB repair, especially Next, the cells were washed with 3% BSA in PBS. In the case of antibodies directed against CPDs (TDM2) (Mori et al., 1991) or 6-4PPs (6-4-M-2) (Mori et al., 1991) since it has recently been effectively applied in mammalian cells were treated with 0.07 M NaOH in PBS for 5 minutes at room temperature to cells (Rodrigue et al., 2006). However, the study of denature the DNA. Next, the cells were washed three times with P-buffer (0.1% accumulation kinetics of DSB repair factors may be more Triton X-100 in PBS) and washed once using I-buffer (0.1% glycine, 1% BSA in PBS). Then, cells were incubated with primary antibodies (diluted in I-buffer) for complicated with this method because the timing of the activity 1 hour at 20°C for detection of protein epitopes or 12 hours at 4°C for detection of of restriction enzymes is difficult to control. DNA lesions. The rabbit anti-H2AX (Ser138) antibody was from Upstate Biotechnology (Charlottesville, VA, USA). After incubation, cells were washed three times using P-buffer, once using I-buffer, and incubated for 1 hour at 20°C Conclusion with secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 594 (or We have shown that most presently available and widely used multiple antibodies for double staining) diluted in I-buffer. Next, cells were washed laser-assisted DNA damaging methods induce a wide response three times using P-buffer, once with PBS and embedded in Vectashield (Vector Journal of Cell Science Laser-induced DNA damage 2739 Laboratory). The rabbit anti-PKcs antibody was a kind gift from D. Chen (Chan of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo. Mol. et al., 2002). The TUNEL staining method was acquired from Roche Applied Cell 10, 1163-1174. Houtsmuller, A. B., Rademakers, S., Nigg, A. L., Hoogstraten, D., Hoeijmakers, J. Science, Penzberg Germany (Cat. No. 12156792910). PARP-1 accumulation was H. J. and Vermeulen, W. (1999). Action of DNA repair endonuclease ERCC1/XPF detected with anti-poly(ADP-ribose) polymerase-1 (human) polyclonal antibody in living cells. Science 284, 958-961. (ALX-210-895) from Alexis (Breda, The Netherlands). Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. 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References