Abstract
Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end-joining (NHEJ) in higher eukaryotes, and have helped to characterize several components including DNA-PKcs, Ku, DNA ligase IV, and XRCC4. There is evidence, however, that additional factors involved in NHEJ remain to be characterized. 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 double-strand breaks (DSBs) induced in plasmid vs 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. Here, we describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for preparation of cell extract and the preparation of the substrate DNA (“naked” DNA or nuclei).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Thompson, L. H. and Limoli, C. L. (2003) Origin, recognition, signaling, and repair of DNA double-strand breaks in mammalian cells. Eurekah.com and Kluwer Academic/Plenum, New York, NY.
Jackson, S. P. (2002) Sensing and repairing DNA double-strand breaks. Carcinogenesis 23, 687–696.
Valerie, K. and Povirk, L. F. (2003) Regulation and mechanisms of mammalian double-strand break repair. Oncogene 22, 5792–5812.
Iliakis, G., Wang, H., Perrault, A. R., et al. (2004) Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet. Genome Res. 104, 14–20.
Baumann, P. and West, S. C. (1998) DNA end-joining catalyzed by human cellfree extracts. Proc. Natl. Acad. Sci. USA 95, 14,066–14,070.
Udayakumar, D., Bladen, C. L., Hudson, F. Z., and Dynan, W. S. (2003) Distinct pathways of nonhomologous end-joining that are differentially regulated by DNA-dependent protein kinase-mediated phosphorylation. J. Biol. Chem. 278, 41,631–41,635.
Huang, J. and Dynan, W. S. (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., et al. (2001) Efficient rejoining of radiationinduced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group. Oncogene 20, 2212–2224.
Wang, H., Perrault, A. R., Takeda, Y., Qin, W., Wang, H. and 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., and 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. and 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. and 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., and 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., and 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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Iliakis, G., Cheong, N. (2006). In Vitro Rejoining of Double-Strand Breaks in Genomic DNA. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:095
Download citation
DOI: https://doi.org/10.1385/1-59259-973-7:095
Publisher Name: Humana Press
Print ISBN: 978-1-58829-513-2
Online ISBN: 978-1-59259-973-8
eBook Packages: Springer Protocols