Published online 12 April 2010 Nucleic Acids Research, 2010, Vol. 38, No. 12 e129 doi:10.1093/nar/gkq226 A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks Keiji Suzuki*, Motohiro Yamauchi, Yasuyoshi Oka, Masatoshi Suzuki and Shunichi Yamashita Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan Received August 27, 2009; Revised March 16, 2010; Accepted March 18, 2010 ABSTRACT role in maintaining genome integrity (1–5). Upon irradi- An ataxia-telangiectasia mutated (ATM)-dependent ation, autophosphorylation and monomerization of DNA damage signal is amplified through the inter- ATM proteins occur, and activated ATM phosphorylates various downstream mediators and effectors, such as action of various factors, which are recruited to the histone H2AX, MDC1, 53BP1 and NBS1. A proper chromatin regions with DNA double-strand breaks. ATM-dependent DNA damage response requires amplifi- Spatial and temporal regulation of such factors cation of the damage signal by recruiting the factors to the is analysed by fluorescence microscopy in combin- site of chromatin with the aid of histone H2AX phosphor- ation with laser micro-irradiation. Here we describe ylation (6,7). The recruited factors create discrete foci in the a novel and simple technique for micro-irradiation nuclei, which are detectable under fluorescence microscopy that does not require a laser source. Cells were (8). These foci are often called ionizing radiation-induced labelled with BrdU for 48–72 h, covered with foci (IRIF). The physiological importance of IRIF forma- porous polycarbonate membranes, and exposed to tion has been demonstrated by various studies, in which the UVC. All BrdU-labelled cells showed localized foci of cells lacking IRIF factors display a compromised DNA phosphorylated ATM, phosphorylated histone damage response, as evidenced by deficiencies in cell-cycle arrest and DNA repair (1,2,5,9–11). H2AX, MDC1 and 53BP1 upon irradiation, showing Activated ATM mediates the phosphorylation of serine that these foci were induced irrespective of the or threonine residues, which create specific docking sites cell-cycle phase. They were also detectable in nu- for proteins harbouring FHA and BRCT domains (12). In cleotide excision repair-defective XPA cells labelled particular, phosphorylation of histone H2AX at serine 139 with BrdU, indicating that the foci did not reflect an is the primary modification, which is essential for persist- excision repair-related process. Furthermore, an ent recruitment of IRIF factors (13). Furthermore, re- ATM-specific inhibitor significantly attenuated cruited proteins, including MDC1, NBS1, MRE11 and the foci formation, and disappearance of the foci 53BP1, are also targets for ATM-dependent phosphoryl- was significantly abrogated in non-homologous ation, which is required for the sequential protein–protein end-joining-defective cells. Thus, it can be con- interactions involved in IRIF formation (14–19). Thus, cluded that micro-irradiation generated DNA analyses of the dynamics of both recruitment and phos- phorylation of the IRIF factors are indispensable for a double-strand breaks in BrdU-sensitized cells. The comprehensive understanding of DNA damage response. present technique should accelerate research in the So far, the foci of phosphorylated ATM and its down- fields of DNA damage response, DNA repair and stream factors have been visualized by fluorometric assays DNA recombination, as it provides more chances using phospho-specific antibodies. Recruitment of MDC1 to perform micro-irradiation experiments without and 53BP1 into the foci is also demonstrated with specific any specific equipment. antibodies. However, some DNA damage response factors, like Ku and DNA–PKcs proteins, have never been found to form foci after conventional irradiation INTRODUCTION (20), while phosphorylated DNA–PKcs formed foci An ataxia-telangiectasia mutated (ATM)-dependent cellu- (21,22). It can be postulated that the number of such lar response to DNA double-strand breaks plays a pivotal DNA repair proteins locally accumulated at the site of *To whom correspondence should be addressed. Tel: +81 95 819 7116; Fax: +81 95 819 7117; Email: [email protected] ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e129 Nucleic Acids Research, 2010, Vol. 38, No. 12 PAGE 2 OF 11 one DNA double-strand break is not sufficient for the de- cells were cultured in a-MEM supplemented with 10% tection by immunofluorescence technique. Therefore, to fetal bovine serum (TRACE Bioscience PTY Ltd., circumvent the problem, localized laser micro-irradiation Australia). XPEMB-1 (JCRB0325) and XP2OS of subnuclear regions in combination with halogenated (KURB1007) cells were obtained from Japanese thymidine analogues has been developed and applied in Collection of Research Bioresources (JCRB). recent studies (8,20,23–25). Exponentially growing cells were plated onto sterilized Since the first study using an ultraviolet A (UVA) laser 22 Â 22 mm cover slips at a density of 5 Â 104 cells per in combination with BrdU was reported, the UVA lasers slip, and the cells were incubated for overnight at 37C of different wavelengths have been used to investigate the in a 5% CO2 incubator. For BrdU labelling, the culture dynamics of DNA damage response and repair factors medium was replaced by a new medium containing 10 mM (8,14,20,23–25). Moreover, green and near-infrared BrdU, and they were incubated for further 48–72 h. The lasers, which do not require DNA sensitization, were ATM kinase activity was inhibited by a specific inhibitor, used in some studies (26–28). Recently, a comparative KU55933 and 40 mM of KU55933 was administrated study discussed mechanisms of DNA damage induction 30 min before UVC-irradiation. Immediately after irradi- by different laser micro-irradiation systems (29). From ation, a fresh medium containing 40 mM of KU55933 was these studies, the usefulness of laser micro-irradiation fed, and the cells were cultured at 37 C in a 5% CO2 in- has already been proved. However, to perform these ex- cubator until they were fixed. periments requires the specific devices, which are the laser sources. Micro-irradiation While UVA lamps have been used for creating DNA Cells were washed with PBS twice, and three-fourth of the double-strand breaks (30), it is impossible to generate cells were covered by a polycarbonate micro-pore localized DNA damage in subnuclear regions without membrane (Isopore membrane, Millipore, Tokyo). Then, any focusing units. In this report, we have developed a cells were exposed to UVC light from germicidal lamp novel and simple micro-irradiation technique by (GL-15, TOSHIBA, Tokyo). Immediately after irradiating cells through micro-pore membranes, whose exposure, a fresh BrdU-free medium was fed, and the application has been reported elsewhere (31–33). In cells were cultured at 37 C in a 5% CO2 incubator for addition, we utilized UVC light from a germicidal lamp, various time before fixation. The dose rate was 1 J/m2 because UVC is significantly more effective than UVA on per second, which was measured by a UV dosimeter photosensitization of BrdU in DNA (30,34). Cells were (UVR-1, TOPCON, Tokyo). labelled with BrdU for 48–72 h, covered by micro-pore membranes, and exposed to UVC light in order to Immunofluorescence induce photochemical events leading to DNA double-strand break. After 1-h incubation, the cells were Cells cultured on coverslips were fixed with 4% formalde- fixed and stained with antibody recognizing hyde for 10 min, permeabilized with 0.5% Triton X-100 phosphorylated histone H2AX at serine 139, whose phos- for 5 min, and were washed extensively with phorylation has been treated as a reliable biochemical phosphate-buffered saline (PBS). Fixation and permeabil- marker for DNA double-strand breaks (16,35,36). We ization were performed on ice. The primary antibodies have successfully created localized DNA double-strand were diluted in 100 ml of TBS–DT (20 mM Tris–HCl, breaks independent of DNA replication and unscheduled pH7.6, 137 mM NaCl, containing 50 mg/ml skim milk DNA synthesis caused by nucleotide excision repair. and 0.1% Tween-20), and the antibodies were applied Activation of ATM and recruitment of DNA damage on the coverslips. The samples were incubated for 2 h in checkpoint factors including MDC1 and 53BP1 were a humidified CO2 incubator at 37 C. Then, the primary also observed. antibodies were washed with PBS, and Alexa488-labelled Our novel technique is a simple and undemanding anti-mouse or Alexa594-labelled anti-rabbit IgG method for generating localized DNA double-strand antibodies (Molecular Probes, Inc., OR) were added. breaks. It also obviates the need for a true radiation The coverslips were incubated for 1 h in a humidified irradiator to generate DNA double-strand breaks. Thus, CO2 incubator at 37 C, washed with PBS, and counter- the method should accelerate research in the fields of stained with 0.1 mg/ml of DAPI. The samples were DNA damage response, DNA repair and DNA recombin- examined with a F3000B fluorescence microscope (Leica, ation without the