A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks.
An ataxia-telangiectasia mutated (ATM)-dependent DNA damage signal is amplified through the interaction of various factors, which are recruited to the chromatin regions with DNA double-strand breaks. Spatial and temporal regulation of such factors is analysed by fluorescence microscopy in combination with laser micro-irradiation. Here we describe a novel and simple technique for micro-irradiation that does not require a laser source. Cells were labelled with BrdU for 48-72 h, covered with porous polycarbonate membranes, and exposed to UVC. All BrdU-labelled cells showed localized foci of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 upon irradiation, showing that these foci were induced irrespective of the cell-cycle phase. They were also detectable in nucleotide excision repair-defective XPA cells labelled with BrdU, indicating that the foci did not reflect an excision repair-related process. Furthermore, an ATM-specific inhibitor significantly attenuated the foci formation, and disappearance of the foci was significantly abrogated in non-homologous end-joining-defective cells. Thus, it can be concluded that micro-irradiation generated DNA double-strand breaks in BrdU-sensitized cells. The present technique should accelerate research in the fields of DNA damage response, DNA repair and DNA recombination, as it provides more chances to perform micro-irradiation experiments without any specific equipment.
Suzuki
Keiji
K
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. kzsuzuki@net.nagasaki-u.ac.jp
Yamauchi
Motohiro
M
Oka
Yasuyoshi
Y
Suzuki
Masatoshi
M
Yamashita
Shunichi
S
D000818:Animals; D001973:Bromodeoxyuridine; D002478:Cells, Cultured; D006224:Cricetinae; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D014466:Ultraviolet Rays
D008567:Membranes, Artificial; D001973:Bromodeoxyuridine
Cold Spring Harb Symp Quant Biol. 2005;70:99-109
16869743
Radiat Res. 2005 Oct;164(4 Pt 2):518-22
16187760
Nature. 2003 Jan 30;421(6922):499-506
12556884
Chromosoma. 2005 Aug;114(3):146-54
15988581
J Biol Chem. 2001 Dec 21;276(51):47759-62
11673449
J Cell Biol. 2000 Jul 24;150(2):283-91
10908572
Mol Cell. 2007 Dec 14;28(5):739-45
18082599
Mol Cell. 2006 Jan 20;21(2):187-200
16427009
J Invest Dermatol. 2001 Nov;117(5):1156-61
11710927
Nat Rev Cancer. 2008 Dec;8(12):957-67
19005492
Mol Cell. 2001 Jul;8(1):213-24
11511374
Science. 1966 Jul 22;153(3734):379-86
5328566
Curr Biol. 2000 Jul 27-Aug 10;10(15):886-95
10959836
Prog Nucleic Acid Res Mol Biol. 2005;79:183-235
16096029
Science. 2007 May 25;316(5828):1160-6
17525332
EMBO Rep. 2001 Nov;2(11):1013-7
11713193
Nucleic Acids Res. 2009 May;37(9):e68
19357094
DNA Repair (Amst). 2008 Mar 1;7(3):530-43
18191622
Eur Biophys J. 2009 Jul;38(6):721-8
19495740
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):855-61
15279770
Radiat Res. 2004 May;161(5):517-27
15161372
Photochem Photobiol. 1989 Jun;49(6):805-19
2672059
Trends Cell Biol. 2009 Nov;19(11):617-29
19819145
Radiat Prot Dosimetry. 2006;122(1-4):147-9
17132661
Mutagenesis. 2003 Sep;18(5):411-6
12960408
J Cell Sci. 2007 Aug 1;120(Pt 15):2731-40
17646676
Trends Cell Biol. 2009 May;19(5):207-17
19342239
Nucleic Acids Res. 2007;35(9):2913-23
17439963
Nat Rev Mol Cell Biol. 2009 Apr;10(4):243-54
19277046
Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9891-6
16788066
Nucleic Acids Res. 2007;35(22):7665-75
17982172
Methods Cell Biol. 2004;75:355-73
15603433
Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3172-7
19221031
J Biol Chem. 2002 Nov 22;277(47):45149-53
12228239
Nat Rev Cancer. 2005 Jul;5(7):564-73
16069818
Hum Genet. 1980;54(1):107-10
6993338
Cell Cycle. 2007 Oct 1;6(19):2366-70
17881893
DNA Repair (Amst). 2006 May 10;5(5):534-43
16531125
Oncogene. 2007 Dec 10;26(56):7773-9
18066090
Nat Rev Cancer. 2003 Mar;3(3):155-68
12612651
J Cell Biol. 2006 Apr 24;173(2):195-206
16618811
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1219-25
15279810
Nat Cell Biol. 2003 Mar;5(3):255-60
12598907
Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13738-43
15365186
Biophys J. 1978 Dec;24(3):645-56
367461
Biochem Soc Trans. 2009 Aug;37(Pt 4):897-904
19614615
J Biol Chem. 2005 Apr 15;280(15):14709-15
15677476
Radiat Res. 2008 Apr;169(4):437-46
18363429
DNA Repair (Amst). 2004 Aug-Sep;3(8-9):959-67
15279782
Nat Rev Mol Cell Biol. 2008 Dec;9(12):958-70
19023283
Science. 2004 Jan 2;303(5654):92-5
14704429
Science. 2001 Nov 23;294(5547):1713-6
11721054
Nat Rev Mol Cell Biol. 2008 Oct;9(10):759-69
18813293
EMBO J. 2006 Dec 13;25(24):5775-82
17124492
Radiat Res. 2006 May;165(5):499-504
16669703
EMBO Rep. 2003 Dec;4(12):1144-9
14618160
J Cell Sci. 2007 Mar 15;120(Pt 6):1104-12
17327276
Genes Dev. 2002 Sep 15;16(18):2333-8
12231622
J Cell Biol. 1999 Sep 6;146(5):905-16
10477747
DNA Repair (Amst). 2008 Mar 1;7(3):405-17
18248856
Nucleic Acids Res. 2008 Oct;36(17):5678-94
18772227
Radiat Res. 1993 May;134(2):160-9
7683818
Nature. 2003 Jan 23;421(6921):436-40
12540918
Mutat Res. 1994 May 1;307(1):15-23
7513792