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DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay
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  • Journal title : Molecules and Cells
  • Volume 39, Issue 3,  2016, pp.204-210
  • Publisher : Korea Society for Molecular and Cellular Biology
  • DOI : 10.14348/molcells.2016.2206
 Title & Authors
DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay
Park, Sojin; Choi, Seoyun; Ahn, Byungchan;
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 Abstract
DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.
 Keywords
Caenorhabditis elegans;comet assay;DNA damage;DNA repair;DNA strand break;
 Language
English
 Cited by
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The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans, Environmental Toxicology and Pharmacology, 2016, 45, 356  crossref(new windwow)
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 References
1.
Adamo, A., Montemauri, P., Silva, N., Ward, J.D., Boulton, S.J., and La, V.A. (2008). BRC-1 acts in the inter-sister pathway of meiotic double-strand break repair. EMBO Rep. 9, 287-292. crossref(new window)

2.
Ahmed, S., and Hodgkin, J. (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159-164. crossref(new window)

3.
Alpi, A., Pasierbek, P., Gartner, A., and Loidl, J. (2003). Genetic and cytological characterization of the recombination protein RAD-51 in Caenorhabditis elegans. Chromosoma 112, 6-16. crossref(new window)

4.
Balajee, A.S., and Geard, C.R. (2004). Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks. Exp. Cell Res. 300, 320-334. crossref(new window)

5.
Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M.B., Bartek, J., and Lukas, J. (2006). Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173, 195-206. crossref(new window)

6.
Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A., and Vidal, M. (2004). BRCA1/BARD1 orthologs required for DNA repair in Caenorhabditis elegans. Curr. Biol. 14, 33-39. crossref(new window)

7.
Broude, N.E., and Budowsky, E.I. (1973). The reaction of glyoxal with nucleic acid components. V. Denaturation of DNA under the action of glyoxal. Biochim. Biophys. Acta 294, 378-384. crossref(new window)

8.
Chen, B.Y., Huang, C.H., Lin, Y.H., Huang, C.C., Deng, C.X., and Hsu, L.C. (2014). The K898E germline variant in the PP1-binding motif of BRCA1 causes defects in DNA Repair. Sci. Rep. 4, 5812.

9.
Chin, G.M., and Villeneuve, A.M. (2001). C. elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint. Genes Dev. 15, 522-534. crossref(new window)

10.
Craig, A.L., Moser, S.C., Bailly, A.P., and Gartner, A. (2012). Methods for studying the DNA damage response in the Caenorhabditis elegans germ line. Methods Cell Biol. 107, 321-352. crossref(new window)

11.
Essers, J., Houtsmuller, A.B., van, V.L., Paulusma, C., Nigg, A.L., Pastink, A., Vermeulen, W., Hoeijmakers, J.H., and Kanaar, R. (2002). Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. EMBO J. 21, 2030-2037. crossref(new window)

12.
Garcia-Muse, T., and Boulton, S.J. (2005). Distinct modes of ATR activation after replication stress and DNA double-strand breaks in Caenorhabditis elegans. EMBO J. 24, 4345-4355. crossref(new window)

13.
Godard, T., Deslandes, E., Sichel, F., Poul, J.M., and Gauduchon, P. (2002). Detection of topoisomerase inhibitor-induced DNA strand breaks and apoptosis by the alkaline comet assay. Mutat. Res. 520, 47-56. crossref(new window)

14.
Hofmann, E.R., Milstein, S., Boulton, S.J., Ye, M., Hofmann, J.J., Stergiou, L., Gartner, A., Vidal, M., and Hengartner, M.O. (2002). Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis. Curr. Biol. 12, 1908-1918. crossref(new window)

15.
Hyun, M., Lee, J., Lee, K., May, A., Bohr, V.A., and Ahn, B. (2008). Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans. Nucleic Acids Res. 36, 1380-1389.

16.
Hyun, M., Park, S., Kim, E., Kim, D.H., Lee, S.J., Koo, H.S., Seo, Y.S., and Ahn, B. (2012). Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1. Biochemistry 51, 1336-1345. crossref(new window)

17.
Kalogeropoulos, N., Christoforou, C., Green, A.J., Gill, S., and Ashcroft, N.R. (2004). chk-1 is an essential gene and is required for an S-M checkpoint during early embryogenesis. Cell Cycle 3, 1196-1200.

18.
Langerak, P., and Russell, P. (2011). Regulatory networks integrating cell cycle control with DNA damage checkpoints and double-strand break repair. Philos. Trans. R. Soc. Lond B Biol. Sci. 366, 3562-3571. crossref(new window)

19.
Lankinen, M.H., Vilpo, L.M., and Vilpo, J.A. (1996). UV- and gamma-irradiation-induced DNA single-strand breaks and their repair in human blood granulocytes and lymphocytes. Mutat. Res. 352, 31-38. crossref(new window)

20.
Lee, S.J., Gartner, A., Hyun, M., Ahn, B., and Koo, H.S. (2010). The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks. PLoS Genet. 6, e1000801. crossref(new window)

21.
Lemmens, B.B., and Tijsterman, M. (2011). DNA double-strand break repair in Caenorhabditis elegans. Chromosoma 120, 1-21. crossref(new window)

22.
MacQueen, A.J., and Villeneuve, A.M. (2001). Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev. 15, 1674-1687. crossref(new window)

23.
Meza, J.E., Brzovic, P.S., King, M.C., and Klevit, R.E. (1999). Mapping the functional domains of BRCA1. Interaction of the ring finger domains of BRCA1 and BARD1. J. Biol. Chem. 274, 5659-5665. crossref(new window)

24.
Miyazaki, T., Bressan, D.A., Shinohara, M., Haber, J.E., and Shinohara, A. (2004). In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J. 23, 939-949. crossref(new window)

25.
Moynahan, M.E., Chiu, J.W., Koller, B.H., and Jasin, M. (1999). Brca1 controls homology-directed DNA repair. Mol. Cell 4, 511-518. crossref(new window)

26.
Nakada, S., Yonamine, R.M., and Matsuo, K. (2012). RNF8 regulates assembly of RAD51 at DNA double-strand breaks in the absence of BRCA1 and 53BP1. Cancer Res. 72, 4974-4983. crossref(new window)

27.
Olive, P.L. (2011). Retention of gammaH2AX foci as an indication of lethal DNA damage. Radiother. Oncol. 101, 18-23. crossref(new window)

28.
Olive, P.L., and Banath, J.P. (2006). The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23-29. crossref(new window)

29.
Olive, P.L., Banath, J.P., and Durand, R.E. (1990). Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat. Res. 122, 86-94. crossref(new window)

30.
Pachkowski, B., and Nakamura, J. (2013). A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks. Methods Mol. Biol. 1054, 133-143. crossref(new window)

31.
Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., and Bonner, W.M. (2000). A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886-895. crossref(new window)

32.
Petalcorin, M.I., Galkin, V.E., Yu, X., Egelman, E.H., and Boulton, S.J. (2007). Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2. Proc. Natl. Acad. Sci. USA 104, 8299-8304. crossref(new window)

33.
Polanowska, J., Martin, J.S., Garcia-Muse, T., Petalcorin, M.I., and Boulton, S.J. (2006). A conserved pathway to activate BRCA1- dependent ubiquitylation at DNA damage sites. EMBO J. 25, 2178-2188. crossref(new window)

34.
Pommier, Y. (2006). Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer 6, 789-802. crossref(new window)

35.
Powell, S.N., and Kachnic, L.A. (2003). Roles of BRCA1 and BRCA2 in homologous recombination, DNA replication fidelity and the cellular response to ionizing radiation. Oncogene 22, 5784-5791. crossref(new window)

36.
Ryan, A.J., Squires, S., Strutt, H.L., and Johnson, R.T. (1991). Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. Nucleic Acids Res. 19, 3295-3300. crossref(new window)

37.
Scully, R., Ganesan, S., Vlasakova, K., Chen, J., Socolovsky, M., and Livingston, D.M. (1999). Genetic analysis of BRCA1 function in a defined tumor cell line. Mol. Cell 4, 1093-1099. crossref(new window)

38.
Singh, N.P., and Khan, A. (1995). Acetaldehyde: genotoxicity and cytotoxicity in human lymphocytes. Mutat. Res. 337, 9-17. crossref(new window)

39.
Sobkowiak, R., and Lesicki, A. (2009). Genotoxicity of nicotine in cell culture of Caenorhabditis elegans evaluated by the comet assay. Drug Chem. Toxicol. 32, 252-257. crossref(new window)

40.
Tarsounas, M., Davies, A.A., and West, S.C. (2004). RAD51 localization and activation following DNA damage. Philos. Trans. R. Soc. Lond B Biol. Sci. 359, 87-93. crossref(new window)

41.
van Veelen, L.R., Cervelli, T., van de Rakt, M.W., Theil, A.F., Essers, J., and Kanaar, R. (2005). Analysis of ionizing radiation-induced foci of DNA damage repair proteins. Mutat. Res. 574, 22-33. crossref(new window)

42.
Wicky, C., Alpi, A., Passannante, M., Rose, A., Gartner, A., and Muller, F. (2004). Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line. Mol. Cell Biol. 24, 5016-5027. crossref(new window)