In situ visualization of DNA double-strand break repair in human fibroblasts

Science

Volumn 280, Issue 5363, 1998, Pages 590-592

  Nelms, Benjamin E ^a   Maser, Richard S ^a   MacKay, James F ^b   Lagally, Max G ^b   Petrini, John H J ^a  

^a UNIVERSITY OF WISCONSIN SCHOOL OF MEDICINE AND PUBLIC HEALTH   (United States)

^b University of Wisconsin ^*  (United States)

Author keywords

[No Author keywords available]

Indexed keywords

DOUBLE STRANDED DNA;

ANALYTIC METHOD; ARTICLE; BINDING SITE; CELL ULTRASTRUCTURE; DNA REPAIR; DNA STRAND BREAKAGE; FIBROBLAST; HUMAN; HUMAN CELL; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN LOCALIZATION;

BROMODEOXYURIDINE; CELL LINE; CELL NUCLEUS; DNA; DNA DAMAGE; DNA REPAIR; DNA-BINDING PROTEINS; FIBROBLASTS; FLUORESCEIN-5-ISOTHIOCYANATE; FLUORESCENT ANTIBODY TECHNIQUE; HUMANS; MICROSCOPY, CONFOCAL; RAD51 RECOMBINASE;

EID: 0032562595     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5363.590     Document Type: Article

References (49)

1. P. Hozak, A. B. Hassan, D. A. Jackson, P. R. Cook, Cell 73, 361 (1993); J. Strouboulis and A. P. Wolffe, J. Cell Sci. 109, 1991 (1996).
2. P. Hozak, A. B. Hassan, D. A. Jackson, P. R. Cook, Cell 73, 361 (1993); J. Strouboulis and A. P. Wolffe, J. Cell Sci. 109, 1991 (1996).
3. R. S. Maser, K. J. Monsen, B. E. Nelms, J. H. J. Petrini, Mol. Cell. Biol. 17, 6087 (1997).
4. T. Haaf, E. I. Golub, G. Reddy, C. M. Radding, D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 92, 2298 (1995).
5. R. Scully et al., Cell 90, 425 (1997).
6. M. S. Park et al., Proc. Natl. Acad. Sci. U.S.A. 93, 8368 (1996).
7. J. F. MacKay et al., Med. Phys., in press.
8. B. E. Nelms et al., Radiat. Res., in press.
9. R. P. Virsik-Peuckert, in Radiation-Induced Chromosome Damage in Man, I. Ishihara and M. S. Sasaki, Eds. (Liss, New York, 1983), vol. 4, pp. 51-69.
10. D. T. Goodhead, J. Thacker, R. Cox, Int. J. Radiat, Biol. 63, 543 (1993).
11. 2, 0.2 mM dithiothreitol, 0.005% Triton X-100, 45 nmol of BrdU per milliliter, 250 units of TdT (Promega, Madison, WI)]. Labeling reactions were stopped by rinsing with tris-buffered saline (TBS) [100 mM tris-HCl (pH 7.5) containing 150 mM NaCl] and cells were incubated for 20 min at 37°C in a blocking solution consisting of 2% bovine serum albumin and 0.3% Triton X-100 in TBS, followed by a 30-min incubation at 37°C with 6.25 mg of FITC-anti-BrdU (Becton Dickinson) per milliliter in blocking solution. Cells were washed for 10 min in three changes of fresh TBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (0.1 μg/ml), and mounted in antifade solution [2.3% diazabicyclo{2.2.2}octane, 90% (v/v) glycerol, 10 mM tris-HCl (pH 7.5)].
12. X. Li and Z. Darzynkiewicz, Cell Prolif. 28, 571 (1995).
13. R. Kodym and E. Hurth, Int. J. Radiat. Biol. 68, 133 (1995).
14. Optical sectioning was carried out with a Bio-Rad MRC600 confocal microscope system (using the FITC filter set; ×60, 1.4 numerical aperture objective). Z-series (in the plane perpendicular to the x-ray beam direction) were collected with a step size of 0.5 μm starting from the plating surface (beam proximal). The average cell thickness was 5 to 5.5 μm. The resulting stacks were viewed and contrast enhanced with NIH Image 1.61 (ftp://codon.nih.gov/ pub/nih-image/). Individual sections were then extracted into Photoshop 4.0 (Adobe).
15. B. Nelms, R. Maser, J. Petrini, data not shown.
16. Neutral comet assays (2) were carried out on human 37Lu fibroblasts after partial volume irradiation. The amount of DSBs remaining in cells observed 5 hours after irradiation (mean tail moment 11.5) was comparable to that in unirradiated controls (mean tail moment 12.9) and was less than the amount of DSBs observed 30 min after irradiation (mean tail moment 50.8).
17. S. W. Botchway, D. L. Stevens, M. A. Hill, T. J. Jenner, P. O'Neill, Radiat. Res. 148, 317 (1997).
18. C. Badie et al., ibid. 144, 26 (1995); C. Badie et al., Cancer Res. 55, 1232 (1995).
19. C. Badie et al., ibid. 144, 26 (1995); C. Badie et al., Cancer Res. 55, 1232 (1995).
20. D. R. Leach, Bioessays 16, 893 (1994); G. J. Sharpies and D. R. Leach, Mol. Microbiol. 17, 1215 (1995); J. C. Connelly and D. R. Leach, Genes Cells 1, 285 (1996).
21. D. R. Leach, Bioessays 16, 893 (1994); G. J. Sharpies and D. R. Leach, Mol. Microbiol. 17, 1215 (1995); J. C. Connelly and D. R. Leach, Genes Cells 1, 285 (1996).
22. D. R. Leach, Bioessays 16, 893 (1994); G. J. Sharpies and D. R. Leach, Mol. Microbiol. 17, 1215 (1995); J. C. Connelly and D. R. Leach, Genes Cells 1, 285 (1996).
23. R. Kanaar and J. H. J. Hoeijmakers, Genes Function 1, 165 (1997).
24. J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, F. W. Stahl, Cell 33, 25 (1983); N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12, 563 (1992); E. L. Ivanov, N. Sugawara, C. I. White, J. E. Haber, ibid. 14, 3414 (1994).
25. J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, F. W. Stahl, Cell 33, 25 (1983); N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12, 563 (1992); E. L. Ivanov, N. Sugawara, C. I. White, J. E. Haber, ibid. 14, 3414 (1994).
26. J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, F. W. Stahl, Cell 33, 25 (1983); N. Sugawara and J. E. Haber, Mol. Cell. Biol. 12, 563 (1992); E. L. Ivanov, N. Sugawara, C. I. White, J. E. Haber, ibid. 14, 3414 (1994).
27. J. H. J. Petrini, D. A. Bressan, M. S. Yao, Semin. Immunol. 9, 181 (1997).
28. P. Sung and D. L. Robberson, Cell 82, 453 (1995); P. Baumann, F. Benson, S. C. West, ibid. 87, 757 (1996); R. C. Gupta. L. R. Bazemore, E. I. Golub, C. M. Radding, Proc. Natl. Acad. Sci. U.S.A. 94, 463 (1997).
29. P. Sung and D. L. Robberson, Cell 82, 453 (1995); P. Baumann, F. Benson, S. C. West, ibid. 87, 757 (1996); R. C. Gupta. L. R. Bazemore, E. I. Golub, C. M. Radding, Proc. Natl. Acad. Sci. U.S.A. 94, 463 (1997).
30. P. Sung and D. L. Robberson, Cell 82, 453 (1995); P. Baumann, F. Benson, S. C. West, ibid. 87, 757 (1996); R. C. Gupta. L. R. Bazemore, E. I. Golub, C. M. Radding, Proc. Natl. Acad. Sci. U.S.A. 94, 463 (1997).
31. After treatment with FITC - anti-BrdU (10), cells were washed in phosphate-buffered saline and then blocked and stained with antisera as in (2) except that Lissamine Rhodamine - conjugated antibody to rabbit immunoglobulin (Jackson ImmunoResearch, West Grove, PA) was substituted for FITC-conjugated secondary antisera.
32. G. M. Dolganov et al., Mol. Cell. Biol. 16, 4832 (1996).
33. M. N. Comforth and J. S. Bedford, Science 222, 1141 (1983); B. Nevaldine et al., Radiat. Res. 133, 370 (1993); M. Lobrich, B. Rydberg, P. K. Cooper, Proc. Natl. Acad. Sci. U.S.A. 92, 12050 (1995).
34. M. N. Comforth and J. S. Bedford, Science 222, 1141 (1983); B. Nevaldine et al., Radiat. Res. 133, 370 (1993); M. Lobrich, B. Rydberg, P. K. Cooper, Proc. Natl. Acad. Sci. U.S.A. 92, 12050 (1995).
35. M. N. Comforth and J. S. Bedford, Science 222, 1141 (1983); B. Nevaldine et al., Radiat. Res. 133, 370 (1993); M. Lobrich, B. Rydberg, P. K. Cooper, Proc. Natl. Acad. Sci. U.S.A. 92, 12050 (1995).
36. 37Lu or 180BR fibroblasts were plated and irradiated (70), fixed in 4% paraformaldehyde in 200 mM sucrose containing PBS (pH 7.4) for 5 min, and then permeabilized for 5 min (4). Cells were incubated in blocking solution and then treated with anti-hMre11 or anti-hRad51 or double-labeled with anti-hRad51 plus Texas Red - conjugated anti-hMre11 (2). Images were captured under epifluorescence with a cooled charge-coupled device camera and images were processed using Photoshop 4.0 (Adobe).
37. A. Yamamoto et al., Mol. Gen. Genet. 251, 1 (1996); J. Flygare, F. Benson, D. Hellgren, Biochim. Biophys. Acta 1312, 231 (1996).
38. A. Yamamoto et al., Mol. Gen. Genet. 251, 1 (1996); J. Flygare, F. Benson, D. Hellgren, Biochim. Biophys. Acta 1312, 231 (1996).
39. R. Cox, J. Thacker, D. T. Goodhead, Int. J. Radiat. Biol. 31, 561 (1977); D. T. Goodhead, J. Thacker, R. Cox, ibid. 36, 101 (1979).
40. R. Cox, J. Thacker, D. T. Goodhead, Int. J. Radiat. Biol. 31, 561 (1977); D. T. Goodhead, J. Thacker, R. Cox, ibid. 36, 101 (1979).
41. R. H. Singer and M. R. Green, Cell 91, 291 (1997).
42. W. G. Nelson and M. B. Kastan, Mol. Cell. Biol. 14, 1815 (1994); L. C. Huang, K. C. Clarkin, G. M. Wahl, Proc. Natl. Acad. Sci. U.S.A. 93, 4827 (1996).
43. W. G. Nelson and M. B. Kastan, Mol. Cell. Biol. 14, 1815 (1994); L. C. Huang, K. C. Clarkin, G. M. Wahl, Proc. Natl. Acad. Sci. U.S.A. 93, 4827 (1996).
44. M. F. Hoekstra, Curr. Opin. Genet. Dev. 7, 170 (1997); S. P. Jackson, Cancer Surv. 28, 261 (1996); P. Jeggo, G. E. Taccioli, S. P. Jackson, Bioessays 17, 949 (1995); D. T. Weaver, Trends Genet. 11, 388 (1995).
45. M. F. Hoekstra, Curr. Opin. Genet. Dev. 7, 170 (1997); S. P. Jackson, Cancer Surv. 28, 261 (1996); P. Jeggo, G. E. Taccioli, S. P. Jackson, Bioessays 17, 949 (1995); D. T. Weaver, Trends Genet. 11, 388 (1995).
46. M. F. Hoekstra, Curr. Opin. Genet. Dev. 7, 170 (1997); S. P. Jackson, Cancer Surv. 28, 261 (1996); P. Jeggo, G. E. Taccioli, S. P. Jackson, Bioessays 17, 949 (1995); D. T. Weaver, Trends Genet. 11, 388 (1995).
47. M. F. Hoekstra, Curr. Opin. Genet. Dev. 7, 170 (1997); S. P. Jackson, Cancer Surv. 28, 261 (1996); P. Jeggo, G. E. Taccioli, S. P. Jackson, Bioessays 17, 949 (1995); D. T. Weaver, Trends Genet. 11, 388 (1995).
48. Y. Shiloh, Annu. Rev. Genet. 31, 635 (1997).
49. We thank our colleagues for materials and advice. Supported by the American Cancer Society (grant NP-918) and the Howard Hughes Medical Institute (J.H.J.P.), NIH grants 5T32GM07133 (R.S.M.) and 5T32GM08349 (B.E.N.), and the Air Force Office of Scientific Research and the National Science Foundation (grant DMR-9212658) (J.F.M. and M.G.L). This work was conducted at the Synchrotron Radiation Center, University of Wisconsin. Paper number 3502 from the University of Wisconsin - Madison Laboratory of Genetics. This paper is dedicated to the memory of our friend and colleague, Tony Bladl.