Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

Jingang Liu^{a, #},
Lu Gong^{a, #},
Changqing Chang^{b, #},
Cong Liu^c,
Jinrong Peng^{b
, ,},
Jun Chen^{a
, ,}

a.
College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China
b.
College of Animal Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China
c.
Developmental and Stem Cell Institute, West China Second University Hospital, Sichuan University, Chengdu 610041, China

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Corresponding author: E-mail address: pengjr@zju.edu.cn (Jinrong Peng); E-mail address: chenjun2009@zju.edu.cn (Jun Chen)

Present address: College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510650, China.

摘要

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Abstract: The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.
- DNA DSB repair /
- NHEJ /
- HR /
- SSA /
- Zebrafish
Present address: College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510650, China.
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参考文献(26)

[1]	Adams, B.R., Hawkins, A.J., Povirk, L.F. et al. ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells Aging (Albany NY), 2 (2010),pp. 582-596
[2]	Akyuz, N., Boehden, G.S., Susse, S. et al. DNA substrate dependence of p53-mediated regulation of double-strand break repair Mol. Cell. Biol., 22 (2002),pp. 6306-6317
[3]	Certo, M.T., Ryu, B.Y., Annis, J.E. et al. Tracking genome engineering outcome at individual DNA breakpoints Nat. Methods, 8 (2011),pp. 671-676
[4]	Chen, J., Ng, S.M., Chang, C. et al. Genes Dev., 23 (2009),pp. 278-290
[5]	Ciccia, A., Elledge, S.J. The DNA damage response: making it safe to play with knives Mol. Cell, 40 (2010),pp. 179-204
[6]	Colleaux, L., D'Auriol, L., Galibert, F. et al. Recognition and cleavage site of the intron-encoded omega transposase Proc. Natl. Acad. Sci. USA, 85 (1988),pp. 6022-6026
[7]	Dudas, A., Chovanec, M. DNA double-strand break repair by homologous recombination Mutat. Res., 566 (2004),pp. 131-167
[8]	Hagmann, M., Adlkofer, K., Pfeiffer, P. et al. Biol. Chem. Hoppe Seyler, 377 (1996),pp. 239-250
[9]	Hagmann, M., Bruggmann, R., Xue, L. et al. Biol. Chem., 379 (1998),pp. 673-681
[10]	Hakem, R. DNA-damage repair; the good, the bad, and the ugly EMBO J., 27 (2008),pp. 589-605
[11]	Hiom, K. Coping with DNA double strand breaks DNA Repair (Amst), 9 (2010),pp. 1256-1263
[12]	Ivanov, E.L., Sugawara, N., Fishman-Lobell, J. et al. Genetics, 142 (1996),pp. 693-704
[13]	Jasin, M. Genetic manipulation of genomes with rare-cutting endonucleases Trends Genet., 12 (1996),pp. 224-228
[14]	Keimling, M., Wiesmuller, L. DNA double-strand break repair activities in mammary epithelial cells – influence of endogenous p53 variants Carcinogenesis, 30 (2009),pp. 1260-1268
[15]	Lieber, M.R., Ma, Y., Pannicke, U. et al. Mechanism and regulation of human non-homologous DNA end-joining Nat. Rev. Mol. Cell Biol., 4 (2003),pp. 712-720
[16]	Mills, K.D., Ferguson, D.O., Essers, J. et al. Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability Genes Dev., 18 (2004),pp. 1283-1292
[17]	Orii, K.E., Lee, Y., Kondo, N. et al. Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development Proc. Natl. Acad. Sci. USA, 103 (2006),pp. 10017-10022
[18]	Pierce, A.J., Johnson, R.D., Thompson, L.H. et al. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells Genes Dev., 13 (1999),pp. 2633-2638
[19]	Porter, G., Westmoreland, J., Priebe, S. et al. Genetics, 143 (1996),pp. 755-767
[20]	Sung, P., Krejci, L., Van, K.S. et al. Rad51 recombinase and recombination mediators J. Biol. Chem., 278 (2003),pp. 42729-42732
[21]	Thoms, K.M., Kuschal, C., Emmert, S. Lessons learned from DNA repair defective syndromes Exp. Dermatol., 16 (2007),pp. 532-544
[22]	Tichy, E.D., Pillai, R., Deng, L. et al. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks Stem Cells Dev., 19 (2010),pp. 1699-1711
[23]	Tichy, E.D., Stambrook, P.J. DNA repair in murine embryonic stem cells and differentiated cells Exp. Cell Res., 314 (2008),pp. 1929-1936
[24]	Weinstock, D.M., Nakanishi, K., Helgadottir, H.R. et al. Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase Methods Enzymol., 409 (2006),pp. 524-540
[25]	Weterings, E., Chen, D.J. The endless tale of non-homologous end-joining Cell Res., 18 (2008),pp. 114-124
[26]	Wilson, J.H., Berget, P.B., Pipas, J.M. Somatic cells efficiently join unrelated DNA segments end-to-end Mol. Cell. Biol., 2 (1982),pp. 1258-1269

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doi: 10.1016/j.jgg.2012.07.009

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Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

Corresponding author: E-mail address: pengjr@zju.edu.cn (Jinrong Peng); E-mail address: chenjun2009@zju.edu.cn (Jun Chen)

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doi: 10.1016/j.jgg.2012.07.009

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Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

Corresponding author: E-mail address: pengjr@zju.edu.cn (Jinrong Peng); E-mail address: chenjun2009@zju.edu.cn (Jun Chen)

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