Abstract
Double-strand breaks (DSB) in genomic DNA are induced by ionizing radiation or radiomimetic drugs but also occur spontaneously during the cell cycle at quite significant frequencies. In vertebrate cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of DSB repair which is able to rejoin two broken DNA termini directly end-to-end irrespective of sequence and structure. Genetic studies in various radiosensitive and DSB repair-deficient cell lines yielded insight into the factors involved in NHEJ. Studies in cell-free systems derived from Xenopus eggs and mammalian cells allowed the dissection of the underlying mechanisms. In the present chapter, we describe a protocol for the preparation of whole cell extracts from mammalian cells and a plasmid-based in vitro assay which permits the easy analysis of the efficiency and fidelity of DSB repair via NHEJ in different cell types.
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Abbreviations
- ATP:
-
Adenosine triphosphate
- BPB:
-
Bromophenol blue
- BSA:
-
Bovine serum albumin
- conc.:
-
Concentration
- dNTP:
-
Deoxy-nucleoside triphosphate
- DSB:
-
Double-strand break
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediamine-tetra-acetate
- EGTA:
-
Ethyleneglycol-bis (β-amino-ethyl) ether-tetra-acetate
- EthBr:
-
Ethidium bromide
- EtOH:
-
Ethanol
- HRR:
-
Homologous recombination repair
- IR:
-
Ionizing radiation
- MOPSO:
-
3-(N-Morpholino)-2-hydroxy-propanesulfonic acid
- NHEJ:
-
Nonhomologous DNA end joining
- O/N:
-
Overnight
- PCV:
-
Packed cell volume
- PMSF:
-
Phenyl-methyl-sulfonyl-fluoride
- ddH2O:
-
Double-distilled water
- RE:
-
Restriction endonuclease
- RT:
-
Room temperature
- SDS:
-
Sodium dodecyl sulfate
- SSA:
-
Single-strand annealing
- UV:
-
Ultra violet
- XC:
-
Xylene cyanol
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Pfeiffer, P., Odersky, A., Goedecke, W., Kuhfittig-Kulle, S. (2014). Analysis of Double-Strand Break Repair by Nonhomologous DNA End Joining in Cell-Free Extracts from Mammalian Cells. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_39
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DOI: https://doi.org/10.1007/978-1-62703-739-6_39
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