Abstract
Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end joining (NHEJ) pathways in higher eukaryotes, and have facilitated the functional characterization of several of its components including DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos, and Artemis. Nevertheless, there is evidence that as of yet uncharacterized repair factors may contribute to the efficiency of NHEJ, for example by modulating the activity of known factors. Also, the discovery of alternative pathways of NHEJ that function as backup to the classical DNA-PK-dependent pathway of NHEJ has added yet another dimension in the set of activities involved. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of DSBs induced in plasmid versus genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: “naked” DNA and DNA organized in chromatin. We describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for the preparation of cell extract and the preparation of the substrate DNA (“naked” DNA or nuclei).
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References
Valerie K, Povirk LF (2003) Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22:5792–5812
Mladenov E, Iliakis G (2011) Induction and repair of DNA double strand breaks: the increasing spectrum of non-homologous end joining pathways. Mutat Res 711:61–7210.1016/j.mrfmmm.2011.02.005
Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:1.1–1.31
Lieber MR (2010) NHEJ and its backup pathways in chromosomal translocations. Nat Struct Mol Biol 17:393–395
Iliakis G, Wang H, Perrault AR, Boecker W, Rosidi B, Windhofer F, Wu W, Guan J, Terzoudi G, Pantelias G (2004) Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet Genome Res 104:14–20
Baumann P, West SC (1998) DNA end-joining catalyzed by human cell-free extracts. Proc Natl Acad Sci U S A 95:14066–14070
Udayakumar D, Bladen CL, Hudson FZ, Dynan WS (2003) Distinct pathways of nonhomologous end joining that are differentially regulated by DNA-dependent protein kinase-mediated phosphorylation. J Biol Chem 278:41631–41635
Huang J, Dynan WS (2002) Reconstruction of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction. Nucleic Acids Res 30:1–8
Wang H, Zeng Z-C, Bui T-A, Sonoda E, Takata M, Takeda S, Iliakis G (2001) Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group. Oncogene 20:2212–2224
Wang H, Perrault AR, Takeda Y, Qin W, Wang H, Iliakis G (2003) Biochemical evidence for Ku-independent backup pathways of NHEJ. Nucleic Acids Res 31:5377–5388
Perrault R, Wang H, Wang M, Rosidi B, Iliakis G (2004) Backup pathways of NHEJ are suppressed by DNA-PK. J Cell Biochem 92:781–794
Labhart P (1999) Nonhomologous DNA end joining in cell-free systems. Eur J Biochem 265:849–861
Ganguly T, Iliakis G (1995) A cell-free assay using cytoplasmic cell extracts to study rejoining of radiation-induced DNA double-strand breaks in human cell nuclei. Int J Radiat Biol 68:447–457
Cheong N, Iliakis G (1997) In vitro rejoining of double strand breaks induced in cellular DNA by bleomycin and restriction endonucleases. Int J Radiat Biol 71:365–375
Cheong N, Perrault R, Iliakis G (1998) In vitro rejoining of DNA double strand breaks: a comparison of genomic-DNA with plasmid-DNA-based assays. Int J Radiat Biol 73:481–493
Cheong N, Okayasu R, Shah S, Ganguly T, Mammen P, Iliakis G (1996) In vitro rejoining of double-strand breaks in cellular DNA by factors present in extracts of HeLa cells. Int J Radiat Biol 69:665–677
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Iliakis, G., Mladenov, E., Cheong, N. (2012). In Vitro Rejoining of Double Strand Breaks in Genomic DNA. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_32
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DOI: https://doi.org/10.1007/978-1-61779-998-3_32
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61779-998-3
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